Detection and treatment of polycystic kidney disease

ABSTRACT

Compositions useful for examining the PKD1 gene are provided. In addition, methods for detecting mutations of the PKD1 gene, which can be associated with autosomal dominant polycystic kidney disease in humans, are provided. Methods for diagnosing a mutant PKD1 gene sequence in a subject also are provided, as are methods of treating a subject having a PKD1-associated disorder.

This application is a continuation application of U.S. application Ser.No. 11/485,062 filed Jul. 11, 2006, now pending; which is a continuationapplication of U.S. application Ser. No. 09/904,968 filed Jul. 13, 2001,now issued as U.S. Pat. No. 7,553,644; which claims the benefit under 35USC §119(e) to U.S. Application Ser. No. 60/283,691 filed Apr. 13, 2001and to U.S. Application Ser. No. 60/218,261 filed Jul. 13, 2000. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

This invention was made with government support under Grant Nos.DK48006, TW05393 and DK02562 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the diagnosis and treatmentof polycystic kidney disease and more specifically to probes and agentsuseful in diagnosing and treating polycystic kidney disease and relateddisorders.

Background Information

Autosomal dominant polycystic kidney disease (ADPKD), also calledadult-onset polycystic kidney disease, is one of the most commonhereditary disorders in humans, affecting approximately one individualin a thousand. The prevalence in the United States is greater than500,000, with 6,000 to 7,000 new cases detected yearly (Striker et al.,Am. J. Nephrol. 6:161-164, 1986; Iglesias et al., Am. J. Kid. Dis.2:630-639, 1983). The disease is considered to be a systemic disorder,characterized by cyst formation in the ductal organs such as kidney,liver, and pancreas, as well as by gastrointestinal, cardiovascular, andmusculoskeletal abnormalities, including colonic diverticulitis, berryaneurysms, hernias, and mitral valve prolapse (Gabow et al., Adv.Nephrol. 18:19-32, 1989; Gabow, New Eng. J. Med. 329:332-342, 1993).

The most prevalent and obvious symptom of ADPKD is the formation ofkidney cysts, which result in grossly enlarged kidneys and a decrease inrenal-concentrating ability. In approximately half of ADPKD patients,the disease progresses to end-stage renal disease, and ADPKD isresponsible for 4-8% of the renal dialysis and transplantation cases inthe United States and Europe (Proc. Eur. Dialysis and Transplant Assn.,Robinson and Hawkins, eds., 17:20, 1981).

Few diagnostics are available for the identification andcharacterization of mutations of the PKD1 gene, which is located onhuman chromosome 16. A major factor contributing to the difficulty inidentifying and characterizing mutations of the PKD1 gene is thatgreater than 70% of the length of the PKD1 gene is replicated onchromosome 16 and elsewhere, resulting in at least six PKD1 homologs.Significantly, the PKD1 homologs share a very high sequence identitywith the PKD1 gene, including sequences having greater than 95% identitywith the PKD1 gene. As such, oligonucleotides that have been examinedfor use as specific probes, or as primers for amplification, of PKD1gene sequences have been found to cross-hybridize with the PKD1homologs, and the inability to identify PKD1 locus specific probes hasprevented accurate analysis of PKD1 gene mutations.

The identification and characterization of PKD1 gene mutations have beenfurther hindered, in part, because transcription of the PKD1 generesults in production of a 14 kilobase (kb) mRNA, which is highlyGC-rich. In addition, unlike the remainder of the PKD1 gene, which isextremely compact (approximately 13.5 kb mRNA coded within approximately30 kb genomic DNA), exon 1 is separated from the rest of the gene by anintron of approximately 19 kb. Thus, previous investigators have simplyplaced the 5′ anchor primer within the first intron and used it as alink to more 3′ sequences. Exon 1 has several other features that havebeen major obstacles to its amplification, including an extremely highGC content (approximately 85%), and the ability to replicate with highfidelity in PKD1 gene homologs. Furthermore, no effective method for DNAbased analysis of PKD1 gene exon 22, which is flanked on both ends byintrons that contain lengthy polypyrimidine tracts. Accordingly, veryfew positions within the replicated segment and flanking exon 22 aresuitable for the design of PKD1-specific primers.

A few oligonucleotides useful for examining regions of the human PKD1gene, have been described. For example, the primer set forth below asSEQ ID NO:11 has been described in U.S. Pat. No. 6,017,717, and theprimer set forth as SEQ ID NO:18 has been described by Watnick et al.(Hum. Mol. Genet. 6:1473-1481, 1997). Also, the primers set forth belowas SEQ ID NOS:9, 10, 49 to 51, and 61 to 105 have been described byWatnick et al. (Am. J. Hum. Genet. 65:1561-1571, 1999). The primers setforth below as SEQ ID NOS: 9 and 10 and SEQ ID NOS: 11 and 12 also weremore recently described by Phakdeekitcharoen et al. (KidneyInternational 58:1400-1412, 2000). In addition, a primer set forth asSEQ ID NO:13 in U.S. Pat. No. 6,071,717 has a nucleotide sequence thatis substantially identical to that set forth below as SEQ ID NO:10, anda primer designated TWR2 by Watnick et al. (Mol. Cell 2:247-251, 1998)has a nucleotide sequence that is substantially identical to that setforth below as SEQ ID NO:12.

Despite the large number of families having diseases associated withPKD1 gene mutations, the potential clinical and scientific impact ofmutation studies, and the availability of a genomic structure, the factthat only a relatively small number of PKD1 mutations have beendescribed demonstrates the relative paucity of data due to thecomplicated genomic structure of the PKD1 gene. Thus, there exists aneed for diagnostic methods suitable for examining the PKD1 gene and foridentifying disorders related to PKD1 gene mutations. The presentinvention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods that allow forthe selective examination of the human PKD1 gene, including thedetection and identification of PKD1 gene mutations. For example, thecompositions of the invention include oligonucleotide primers that areuseful for selectively amplifying a region of a PKD1 gene, but not acorresponding region of a PKD1 homolog. Accordingly, the presentinvention relates to a PKD1 gene specific primer, which can be one of aprimer pair. A primer of the invention includes a 5′ region and adjacentPKD1-specific 3′ region, wherein the 5′ region has a nucleotide sequencethat can hybridize to a PKD1 gene sequence and, optionally, to a PKD1homolog sequence, and the 3′ region has a nucleotide sequence thatselectively hybridizes only to a PKD1 gene sequence, and particularlynot to a PKD1 gene homolog sequence, except that a primer of theinvention does not have a sequence as set forth in SEQ ID NO:11, SEQ IDNO:18, SEQ ID NO:52, or SEQ ID NO:60. A 5′ region of a primer of theinvention generally contains at least about ten contiguous nucleotides,and the 3′ region contains at least one 3′ terminal nucleotide, whereinthe at least one 3′ terminal nucleotide is identical to a nucleotidethat is 5′ and adjacent to the nucleotide sequence of the PKD1 gene towhich the 5′ region of the primer can hybridize, and is different from anucleotide that is 5′ and adjacent to a nucleotide sequence of the PKD1homolog to which the 5′ region of the primer can hybridize. Generally,the primer includes a 5′ region of about 14 to 18 nucleotides and a 3′region of about 2 to 6 nucleotides, particularly about 2 to 4nucleotides. For example, a primer of the invention can have a sequenceas set forth in any of SEQ ID NOS:3 to 10, 12 to 17, 19 to 51 and 61 to113.

The present invention also relates to an isolated mutant PKD1polynucleotide, or an oligonucleotide portion thereof. Thepolynucleotides of the invention are exemplified by mutation of SEQ IDNO:1, which appear to be normal variants that are not associated with aPKD1-associated disorder, for example, a polynucleotide oroligonucleotide that includes nucleotide 474, wherein nucleotide 474 isa T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide 9367,wherein nucleotide 9367 is a T; nucleotide 10143, wherein nucleotide10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C;nucleotide 10255, wherein nucleotide 10255 is a T; or a combinationthereof; and by mutations of SEQ ID NO:1 that are associated with aPKD1-associated disorder, for example, a polynucleotide oroligonucleotide that includes nucleotide 3110 of SEQ ID NO:1, whereinnucleotide 3110 is a C; nucleotide 8298 of SEQ ID NO:1, whereinnucleotide 8298 is a G; nucleotide 9164 of SEQ ID NO:1, whereinnucleotide 9164 is a G; nucleotide 9213 of SEQ ID NO:1, whereinnucleotide 9213 is an A; nucleotide 9326 of SEQ ID NO:1, whereinnucleotide 9326 is a T; nucleotide 10064 of SEQ ID NO:1, whereinnucleotide 10064 is an A; or a combination thereof. The invention alsoprovides a vector containing such a polynucleotide, or anoligonucleotide portion thereof, and provides a host cell containingsuch a polynucleotide or oligonucleotide, or vector.

A PKD1-specific primer of the invention is exemplified by anoligonucleotide that can selectively hybridize to a nucleotide sequencethat flanks and is within about fifty nucleotides of a nucleotidesequence selected from about nucleotides 2043 to 4209; nucleotides 17907to 22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615;nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides37329 to 41258; and nucleotides 41508 to 47320 of SEQ ID NO:1. Theprimer, which can be one of a primer pair, can have a nucleotidesequence substantially identical to any of SEQ ID NOS: 3 to 18, providedthat when the primer is not one of a primer pair, the primer does nothave a sequence as set forth in SEQ ID NO:11, SEQ ID NO:18, SEQ IDNO:52, or SEQ ID NO:60. Accordingly, the present invention furtherrelates to a primer pair that can amplify a portion of a PKD1 gene, forexample, the wild type PKD1 gene set forth as SEQ ID NO:1, wherein theamplification product can include about nucleotides 2043 to 4209;nucleotides 17907 to 22489; nucleotides 22218 to 26363; nucleotides26246 to 30615; nucleotides 30606 to 33957; nucleotides 36819 to 37140;nucleotides 37329 to 41258; nucleotides 41508 to 47320; or a combinationthereof. A primer pair of the invention is useful for performingPKD1-specific amplification of a portion of a PKD1 gene.

Primer pairs of the invention are exemplified by a pair including atleast one forward primer and at least one reverse primer of theoligonucleotides sequences set forth in SEQ ID NOS:3 to 18 or a sequencesubstantially identical thereto. In one embodiment, the primer pairincludes SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ IDNOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113. Alsoprovided are primer pairs useful for performing nested amplification ofa PKD1-specific amplification product of a PKD1 gene, for example, theprimer pairs set forth as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22;SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ IDNOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48;SEQ ID NOS:49 and 50; SEQ ID NOS: 51 and 61; SEQ ID NOS:62 and 63; SEQID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ IDNOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID NOS:76and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81; SEQ ID NOS:82 and83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID NOS:88 and 89;SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ IDNOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ IDNOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and 106; SEQ IDNOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111 and 112. Inanother embodiment, the invention relates to a plurality of primerpairs, which can include two or more primer pairs that are useful forgenerating two or more PKD1-specific amplification products of a PKD1gene; or can include two or more primer pairs that are useful forgenerating a PKD1-specific amplification product of a PKD1 gene and forgenerating a nested amplification product of the PKD1-specificamplification product.

The present invention also relates to a purified mutant PKD1polypeptide, or a peptide portion thereof, comprising an amino acidsequence of a mutant of SEQ ID NO:2. A mutant PKD1 polypeptide, orpeptide portion thereof can be substantially identical to a sequence ofSEQ ID NO:2 and, for example, include amino acid residue 88 of SEQ IDNO:2, wherein residue 88 is a V; residue 967 of SEQ ID NO:2, whereinresidue 967 is an R; residue 2696 of SEQ ID NO:2, wherein residue 2696is an R; residue 2985 of SEQ ID NO:2, wherein residue 2985 is a G;residue 3039 of SEQ ID NO:2, wherein residue 3039 is a C; residue 3285of SEQ ID NO:2, wherein residue 3285 is an I; or residue 3311 of SEQ IDNO:2, wherein residue 3311 is an R; or can include residue 3000 of atruncated mutant PKD1 polypeptide ending at amino acid residue 3000 withrespect to SEQ ID NO:2, wherein residue 3001 is absent (and the mutantPKD1 polypeptide is truncated) due to the presence of a STOP codon inthe encoding mutant PKD1 polynucleotide; or a combination of suchmutations. Also provided is a purified antibody that specifically bindsto a mutant PKD1 polypeptide, or to a peptide thereof.

The present invention further relates to a primer or an oligonucleotideof the invention immobilized to a solid support. In addition, the primeror oligonucleotide can be one of a plurality of primers,oligonucleotides, or a combination thereof, each of which is immobilizedto a solid support. The solid support can be any support, including, forexample, a microchip, in which case, the primers, oligonucleotides, orcombination thereof can be arranged in array, particularly anaddressable array. The primers, oligonucleotides, or combination thereofalso can be degenerate with respect to each other, and specific for awild type PKD1 polynucleotide, a mutant PKD1 polynucleotide, including avariant, or combinations thereof, and, therefore, provide a means formultiplex analysis. Accordingly, the present invention providescompositions comprising one or a plurality of immobilized primers oroligonucleotides of the invention, or combinations thereof.

The present invention also relates to a method of detecting a PKD1polynucleotide in a sample, wherein the PKD1 polynucleotide is a wildtype PKD1 polynucleotide having a sequence as set forth in SEQ ID NO:1,or a mutant PKD1 polynucleotide, which can be a variant PKD1polynucleotide that has a sequence different from SEQ ID NO:1 but is notassociated with a PKD1-associated disorder or can be a mutant PKD1polynucleotide that is associated with a PKD1-associated disorder. Amethod of the invention can be performed, for example, by contactingnucleic acid molecules in a sample suspected of containing a PKD1polynucleotide with at least one primer pair under conditions suitablefor amplification of a PKD1 polynucleotide by the primer pair; andgenerating a PKD1-specific amplification product under said conditions,thereby detecting a PKD1 polynucleotide in the sample. The primer paircan be any primer pair as disclosed herein, for example, a primer pairsuch as SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ IDNOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113; or can bea combination of such primer pairs.

A method of detecting a PKD1 polynucleotide can further include, upongenerating a PKD1-specific amplification product, contacting theamplification product with at least a second primer pair, underconditions suitable for nested amplification of the PKD1-specificamplification product by the second primer pair, and generating a nestedamplification product. The second primer pair can be any primer pairthat can produce a nested amplification product of the PKD1-specificamplification product, for example, a second primer pair such as SEQ IDNOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38;SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ IDNOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51and 61; primer pairs formed using consecutive primers set forth in Table2 as SEQ ID NOS:62 to 96, 113, and 97 to 112; or a combination thereof.

Upon detecting a PKD1 polynucleotide in a sample according to a methodof the invention, an additional step of detecting the presence orabsence of a mutation in an amplification product of the PKD1polynucleotide in the sample as compared to a corresponding nucleotidesequence in SEQ ID NO:1. As such, a method of the invention provides ameans to identify a PKD1 polynucleotide in a sample as a mutant PKD1polynucleotide or a wild type PKD1 polynucleotide, wherein detecting theabsence of a mutation in the amplification product identifies the PKD1polynucleotide in the sample as a wild type PKD1 polynucleotide, andwherein detecting the presence of a mutation in the amplificationproduct identifies the PKD1 polynucleotide in the sample as a mutantPKD1 polynucleotide, which can be a variant PKD1 polynucleotide, or canbe mutant PKD1 polynucleotide associated with a PKD1-associateddisorder, the latter of which are exemplified by a polynucleotide thatis substantially identical to SEQ ID NO:1, and wherein at leastnucleotide 474 is a T; nucleotide 487 is an A; nucleotide 3110 is a C;nucleotide 8298 is a G; nucleotide 9164 is a G; nucleotide 9213 is an A;nucleotide 9326 is a T; nucleotide 9367 is a T; nucleotide 10064 is anA; nucleotide 10143 is a G; nucleotide 10234 is a C; or nucleotide 10255is a T.

The presence or absence of a mutation in an amplification productgenerated according to a method of the invention can be detected anymethod useful for detecting a mutation. For example, the nucleotidesequence of the amplification product can be determined, and can becompared to the corresponding nucleotide sequence of SEQ ID NO:1. Themelting temperature of the amplification product also can be determined,and can be compared to the melting temperature of a corresponding doublestranded nucleotide sequence of SEQ ID NO:1. The melting temperature canbe determined using a method such as denaturing high performance liquidchromatography.

An advantage of a method of the invention is that a large number ofsamples can be examined serially or in parallel. Accordingly, a methodof the invention can be performed with respect to a plurality ofsamples, and can be performed using a high throughput format, forexample, by organizing the samples of a plurality of samples in an arraysuch as in an array is on a microchip. The method can further includedetecting the presence or absence of a mutation in an amplificationproduct of the samples of the plurality of samples, for example, bydetermining the melting temperature of the amplification product andcomparing it to the melting temperature of a corresponding nucleotidesequence of SEQ ID NO:1 using a method such as denaturing highperformance liquid chromatography, or the presence or absence of amutation can be performed using any method useful for such a purpose,for example, matrix-assisted laser desorption time of flight massspectrometry or high throughput conformation-sensitive gelelectrophoresis, each of which is readily adaptable to a high throughputanalysis foimat.

In another embodiment, the presence or absence of a mutation in anamplification product can be detected by contacting the amplificationproduct with the oligonucleotide of the invention, under conditionsuitable for selective hybridization of the oligonucleotide to anidentical nucleotide sequence; and detecting the presence or absence ofselective hybridization of the oligonucleotide to the amplificationproduct. Using such a method detecting the presence of selectivehybridization identifies the PKD1 polynucleotide in the sample as amutant PKD1 polynucleotide, and detecting the absence of selectivehybridization identifies the PKD1 polynucleotide as a wild type PKD1polynucleotide. Where an absence of a mutation is detected, the PKD1polynucleotide in the sample is identified as a wild type PKD1polynucleotide. In comparison, where the presence of a mutation isidentified, the mutant PKD1 polynucleotide so identified can be furtherexamined to determine whether the mutant PKD1 polynucleotide is avariant PKD1 polynucleotide, which is associated with a normal phenotypewith respect to PKD1, for example, where the amplification product has anucleotide sequence substantially identical to SEQ ID NO:1, andincluding C474T, G487A, G4885A; C6058T; G6195A; T7376C; C7696T; G8021A;C9367T, A10143G, T10234C, or a combination thereof, or is a mutant PKD1polynucleotide associated with a PKD1-associated disorder, for example,where the amplification product has a nucleotide sequence substantiallyidentical to SEQ ID NO:1, and including T3110C, G3707A; T6078A; C7433T;T8298G; A9164G; G9213A, C9326T; G10064A; an insertion of GCG betweennucleotides G7535 and A7536; or a combination thereof, each of which isassociated with ADPKD (see Example 2; see, also, Phakdeekitcharoen etal., Kidney International 58:1400-1412, 2000, which is incorporatedherein by reference).

The present invention further relates to a method of detecting thepresence of a mutant PKD1 polynucleotide in a sample. In one embodiment,a method of the invention is performed by amplifying a nucleic acidsequence in a sample suspected of containing a mutant PKD1polynucleotide using a primer pair of the invention, for example, aprimer pair selected from SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ IDNOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and113, thereby obtaining a PKD1-specific amplification product of a PKD1gene sequence; and detecting a mutant PKD1 polynucleotide in theamplification product. The mutant PKD1 nucleotide in the amplificationproduct can be detected using any method useful for detecting a mutationin a polynucleotide, for example, using denaturing high performanceliquid chromatograph. In another embodiment, a method of the inventionis performed by contacting a sample suspected of containing a mutantPKD1 polynucleotide with a probe comprising an isolated polynucleotideof the invention, or an oligonucleotide portion thereof, underconditions such that the probe selectively hybridizes to a mutant PKD1polynucleotide, and detecting specific hybridization of the probe and aPKD1 polynucleotide, thereby detecting the presence of a mutant PKD1polynucleotide sequence in the sample.

The present invention further relates to a method of identifying asubject having or is at risk of having a PKD1-associated disorder. Sucha method can be performed, for example, by contacting nucleic acidmolecules in a sample from a subject with at least one primer pair ofthe invention under conditions suitable for amplification of a PKD1polynucleotide by the primer pair, thereby generating an amplificationproduct; and testing an amplification product for the presence orabsence of a mutation indicative of a PKD1-associated disorder. Asdisclosed herein, the absence of such a mutation identifies the subjectas not having or at risk of the having a PKD1-associated disorder,wherein the presence of such a mutation identifies the subject as havingor is at risk of having a PKD1-associated disorder, for example, ADPKDor acquired cystic disease.

A primer pair useful in a diagnostic method of the invention can includeat least one primer pair selected from SEQ ID NO:3 and 4; SEQ ID NO:5and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12;SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; andSEQ ID NOS:9 and 113. The subject can be any subject having a PKD1 geneand susceptible to a PKD1-associated disorder, including a vertebratesubject, and particularly a mammalian subject such as a cat or a human.In addition, the diagnostic method can be performed in a high throughputformat, thereby allowing the examination of a large number samples in acost-effective manner.

The diagnostic method can further include contacting the amplificationproduct generated as described above with at least a second primer pair,under conditions suitable for nested amplification of the amplificationproduct by a second primer pair, thereby generating a nestedamplification product. The second primer pair can be, for example, aprimer pair selected from SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22;SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ IDNOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48;SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; a primer pair formed usingtwo consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96,113, and 97 to 112 (i.e., SEQ ID NOS: 62 and 63, SEQ ID NOS:64 and 65,and so on); and a combination thereof, in which case, the step oftesting the amplification product for the presence or absence of amutation comprises testing the nested amplification product. It shouldbe recognized that the selection of a primer pair for nestedamplification is based, in part, on the sequence of the PKD1-specificamplification product that is to be used as a template for the nestedamplification, i.e., nested primer pairs are selected such that they canhybridize to a target PKD1-specific amplification product and canamplify the target sequence.

An amplification product can be tested for the presence or absence ofthe mutation, for example, by determining the nucleotide sequence of theamplification product, and comparing it to a corresponding nucleotidesequence of SEQ ID NO:1; by determining the melting temperature of theamplification product, and comparing it to the melting temperature of acorresponding nucleotide sequence of SEQ ID NO:1, for example, using amethod such as denaturing high performance liquid chromatography; or bycontacting the amplification product with an oligonucleotide probecontaining nucleotide 474 of SEQ ID NO:1, wherein nucleotide 474 is a T;nucleotide 487 of SEQ ID NO:1, wherein nucleotide 487 is an A;nucleotide 3110 of SEQ ID NO:1, wherein nucleotide 3110 is a C;nucleotide 8298 of SEQ ID NO:1, wherein nucleotide 8298 is a G;nucleotide 9164 of SEQ ID NO:1, wherein nucleotide 9164 is a G;nucleotide 9213 of SEQ ID NO:1, wherein nucleotide 9213 is an A;nucleotide 9326 of SEQ ID NO:1, wherein nucleotide 9326 is a T;nucleotide 9367 of SEQ ID NO:1, wherein nucleotide 9367 is a T;nucleotide 10064 of SEQ ID NO:1, wherein nucleotide 10064 is an A;nucleotide 10143 of SEQ ID NO:1, wherein nucleotide 10143 is a G;nucleotide 10234 of SEQ ID NO:1, wherein nucleotide 10234 is a C; andnucleotide 10255 of SEQ ID NO:1, wherein nucleotide 10255 is a T, underconditions suitable for selective hybridization of the probe to a mutantPKD1 polypeptide, which can be a normal variant or can be a mutant PKD1polynucleotide associated with a PKD1-associated disorder.

The present invention also relates to a method of diagnosing aPKD1-associated disorder in a subject suspected of having aPKD1-associated disorder. Such a method is performed by amplifying anucleic acid sequence in a sample obtained from the subject using aprimer pair suitable for PKD1-specific amplification of a PKD1 genesequence, for example, a primer pair such as SEQ ID NO:3 and 4; SEQ IDNOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18, orSEQ ID NOS:9 and 113, thereby obtaining a PKD1-specific firstamplification product; and detecting a mutation of a PKD1 gene sequencein the PKD1-specific first amplification product, wherein the mutationis indicative of a PKD1-associated disorder, thereby diagnosing aPKD1-associated disorder in the subject.

In one embodiment, the diagnostic method includes a step of furtheramplifying the first amplification product using a second set of primerpairs to obtain a nested amplification product; and detecting a PKD1gene mutation in the nested amplification product. The second set ofprimer pairs can be any primer pairs useful for amplifying thePKD1-specific first amplification product, including, for example, theprimer pairs exemplified by SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22;SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ IDNOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48;SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; or any of the primer pairsformed using consecutive primers set forth in Table 2 as SEQ ID NOS:62to 96, 113, and 97 to 112.

In another method, the diagnostic method includes a step of contactingthe PKD1-specific first amplification product or second amplificationproduct with a probe comprising an isolated polynucleotide, or anoligonucleotide portion thereof, comprising a mutant of SEQ ID NO:1,under conditions such that the probe can selectively hybridize to amutant PKD1 polynucleotide; and detecting selective hybridization of theprobe to the first amplification product, thereby diagnosing aPKD1-associated disorder in the subject. The probe can be, for example,an oligonucleotide portion of SEQ ID NO:1 that includes one or more ofnucleotide 474 is a T; nucleotide 487 is an A; nucleotide 3110 is a C;nucleotide 8298 is a G; nucleotide 9164 is a G; nucleotide 9213 is an A;nucleotide 9326 is a T; nucleotide 9367 is a T; nucleotide 10064 is anA; nucleotide 10143 is a G; nucleotide 10234 is a C; or nucleotide 10255is a T.

The present invention also relates to a method of detecting the presenceof a mutant PKD1 polypeptide in a sample. Such a method can beperformed, for example, by contacting a sample suspected of containing amutant PKD1 polypeptide with an antibody that specifically binds to amutant PKD1 polypeptide, under conditions which allow the antibody tobind to the mutant PKD1 polypeptide and detecting specific binding ofthe antibody and the mutant PKD1 polypeptide in the sample. Thedetection of an immunocomplex of the antibody and a mutant PKD1polypeptide, for example, indicates the presence of a mutant PKD1polypeptide in the sample. In one embodiment, the method is performed bycontacting a tissue sample from a subject suspected of containing a PKD1polypeptide with the antibody that specifically binds a mutant PKD1polypeptide under conditions that allow the antibody interact with aPKD1 polypeptide and detecting specific binding of the antibody and thePKD1 polypeptide in the tissue.

The present invention further relates to a kit for detecting a mutantPKD1 polynucleotide, which can be a variant PKD1 polynucleotide or amutant PKD1 polynucleotide associated with a PKD1-associated disorder.The kit can contain, for example, a carrier means containing therein oneor more containers wherein a first container contains a nucleotidesequence useful for detecting a wild type or mutant PKD1 polynucleotide.As such, a nucleotide sequence useful in a kit of the invention can bean oligonucleotide comprising at least ten contiguous nucleotides of SEQID NO:1, including at least one of nucleotide 474, wherein nucleotide474 is a T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide3110, wherein nucleotide 3110 is a C; a position corresponding tonucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide 3707,wherein nucleotide 3707 is an A; nucleotide 4168, wherein nucleotide4168 is a T; nucleotide 4885, wherein nucleotide 4885 is an A;nucleotide 5168, wherein nucleotide 5168 is a T; nucleotide 6058,wherein nucleotide 6058 is a T; nucleotide 6078, wherein nucleotide 6078is an A; nucleotide 6089, wherein nucleotide 6089 is a T; nucleotide6195, wherein nucleotide 6195 is an A; nucleotide 6326, whereinnucleotide 6326 is a T; a position corresponding to nucleotides 7205 to7211, wherein nucleotides 7205 to 7211 are deleted; nucleotide 7376,wherein nucleotide 7376 is a C; a nucleotide sequence corresponding tonucleotides 7535 to 7536, wherein a GCG nucleotide sequence is insertedbetween nucleotides 7535 and 7536; nucleotide 7415, wherein nucleotide7415 is a T; nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide7696, wherein nucleotide 7696 is a T; nucleotide 7883, whereinnucleotide 7883 is a T; nucleotide 8021, wherein nucleotide 8021 is anA; a nucleotide sequence corresponding to nucleotide 8159 to 8160,wherein nucleotides 8159 to 8160 are deleted; nucleotide 8298, whereinnucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G;nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326,wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367is a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide10143, wherein nucleotide 10143 is a G; nucleotide 10234, whereinnucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255is a T. A nucleotide sequence useful in a kit of the invention also cancomprise one or both primers of a primer pair, particularly at least aforward primer and a reverse primer as set forth in SEQ ID NOS: 3 to 18;and the kit can further include at least a second primer pair, includinga forward and reverse primer as set forth in SEQ ID NOS: 19 to 51 and 61to 113. In another aspect, the present invention relates to a kitcontaining an antibody that specifically binds to a mutant PKD1polypeptide or peptide portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the genomic structure of the PKD1 gene(SEQ ID NO:1) and the relative position of locus-specific templates andprimers.

FIG. 2 shows the relative position of the BPF6-BPR6 long-range PCRtemplate and the much shorter PKD1-specific exon 28 product, 28F-BPR6.The dashed line below exon 28 identified the long range PCRamplification product that resulted when BPF6, the sequence of which iscommon to the PKD1 gene and to the homologs, was used in combinationwith the homolog-specific primer, BPR6HG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for identifyingpolycystic kidney disease-associated protein-1 (PKD1) gene variants andmutants, and for diagnosing PKD1-associated disorders in a subject.Prior to the present disclosure, the ability to selectively examine theentire PKD1 gene for mutations was precluded due to the high sequencehomology of the PKD1 gene and the PKD1 gene homologs, including thosepresent with the PKD1 gene on human chromosome 16. As disclosed herein,polynucleotide sequences have now been developed that are useful asprobes and primers for examining the entire PKD1 gene. Accordingly, thepresent invention provides polynucleotides, and oligonucleotide portionsthereof, of a PKD1 gene and of PKD1 gene mutants that are useful fordetecting PKD1 mutations, and that can be diagnostic of aPKD1-associated disorder.

Autosomal dominant polycystic kidney disease (ADPKD) exhibits atransmission pattern typical of autosomal dominant inheritance, wheretypically each offspring of an affected individual has a 50% chance ofinheriting the causative gene. Linkage studies indicated that acausative gene is present on the short arm of chromosome 16, near the Iglobin cluster; this locus was designated PKD1 (Reeders et al., Nature,317:542, 1985.) Though other PKD-associated genes exist (for example,PKD2), defects in PKD1 appear to cause ADPKD in about 85-90% of affectedfamilies (Parfrey et al., New Eng. J. Med. 323:1085-1090, 1990; Peterset al., Contrib. Nephrol. 97:128-139, 1992).

The PKD1 gene has been localized to chromosomal position 16p13.3,specifically to an interval of approximately 600 kb between the markersATPL and CMM65 (D16S84). This region is rich in CpG islands that oftenflank transcribed sequences; it has been estimated that this intervalcontains at least 20 genes. The precise location of the PKD1 gene waspinpointed by the finding of an ADPKD family whose affected memberscarry a translocation that disrupts a 14 kb RNA transcript associatedwith this region (European PKD Consortium, Cell, 77:881, 1994).

The genomic structure of the PKD1 gene, which is illustrated in FIG. 1(SEQ ID NO:1; see Appendix A; see, also, GenBank Accession No. L39891,which is incorporated herein by reference), extends over approximately50 kb, contains 46 exons, and is bisected by two large polypyrimidinetracts of approximately 2.5 kb and 0.5 kb, respectively, in introns 21and 22 (indicated by “ . . . CCTCCTCCT . . . ” in FIG. 1). Thereplicated portion of the gene, which begins prior to the 5′UTR and isbelieved to end in exon 34 (FIG. 1; stippled region), coversapproximately two thirds of the 5′ end of the gene and is duplicatedseveral times in a highly similar, transcribed fashion elsewhere in thehuman genome (Germino et al., Genomics 13:144-151, 1992; EuropeanChromosome 16 Tuberous Sclerosis Consortium, 1993, Cell 75:1305-1315).The encoded PKD1 polypeptide is shown as SEQ ID NO:2 (see Appendix A;see, also, GenBank Accession No. P98161, which is incorporated herein byreference). It should be recognized that SEQ ID NO:2 is not the sameamino acid sequence as that shown to be encoded by GenBank Accession No.L39891 (see, also, GenBank AAB59488), presumably due to errors inpredicting the encoded PKD1 polypeptide from the PKD1 gene sequence.Instead, the wild type PKD1 polypeptide sequence is shown in SEQ ID NO:2(GenBank Accession No. P98161).

The present invention provides a PKD1 gene specific primer, which can beone of a primer pair. A primer of the invention includes a 5′ region andadjacent PKD1-specific 3′ region, wherein the 5′ region has a nucleotidesequence that can hybridize to a PKD1 gene sequence or to a PKD1 genesequence and a PKD1 gene homolog sequence, and the 3′ region has anucleotide sequence that selectively hybridizes only to a PKD1 genesequence, and particularly not to a PKD1 gene homolog sequence, exceptthat a primer of the invention does not have a sequence as set forth inSEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. Thus, aprimer of the invention can have a sequence as set forth in any of SEQID NOS:3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112 and 113, as well as asequence that is substantially identical to any of SEQ ID NOS:3 to 51and 61 to 113, provided the sequence comprises a 5′ region that canhybridize to a PKD1 gene sequence or to a PKD1 gene sequence and a PKD1gene homolog sequence, and a 3′ region that selectively hybridizes to aPKD1 gene sequence, but not to a PKD1 gene homolog sequence; andprovided the sequence is not otherwise specifically excluded herein.

As disclosed herein, a primer of the invention can be prepared byaligning SEQ ID NO:1 with the PKD1 gene homologs contained in GenBankAccession Nos. AC002039, AC010488, AC040158, AF320593 and AF320594 (eachof which is incorporated herein by reference; see, also, Bogdanova etal., Genomics 74:333-341, 2001, which is incorporated herein byreference) and identifying regions having potential sequencedifferences, then selecting as PKD1-specific primers those sequencesthat match over at least about ten nucleotides and that have a mismatchat or adjacent to the 3′ terminus of the matched regions (see Example 1;see, also, Phakdeekitcharoen et al., supra, 2000). Such primers arereferred to as “PKD1-specific primers” because, while they can hybridizeto a PKD1 gene and a PKD1 gene homologue, an extension product only canbe generated upon hybridization to a PKD1 gene due to the mismatch ofone or more nucleotides in the 3′ region when the primer hybridizes to aPKD1 gene homologue. Confirmation that a selected oligonucleotide is aPKD1-specific primer can be made using methods as disclosed herein(Example 1) or otherwise known in the art. For example, a simple andstraightforward method for determining that a primer is a PKD1-specificprimer of the invention is to perform a primer extension or anamplification reaction using the putative PKD1-specific primer andtemplates including a PKD1 gene sequence and PKD1 gene homologsequences, and detecting a single extension product or amplificationproduct generated from the PKD1 gene template, but not the PKD1 genehomolog templates. Sequences identified as PKD1-specific primers usingthis or another method can be confirmed by performing various controlexperiments as described by Watnick et al. (supra, 1999), for example,by comparing an amplification product obtained in a cell having a PKD1gene with the products, if any, produced using the radiation hybrid cellline, 145.19, which lacks the PKD1 gene but contains PKD1 gene homologs.

A nucleotide sequence suspected of being useful as a PKD1-specificprimer also can be compared against a human genomic DNA database using,for example, a BLAST search or other algorithm, to confirm that thenucleotide sequence meets the requirements of a PKD1-specific primer asdefined herein. For example, a putative PKD1-specific primer can beexamined at the National Center for Biotechnology Information (NCBI),which can be accessed on the world wide web, by selecting the “Blast”option, thereafter selecting the “Search for short nearly exactmatches,” entering in the sequence to be examined, and, using thedefault search algorithms (word size 7), searching the “nr” database,which include all non-redundant GenBank+EMBL+DDBJ+PDB sequences, but noEST, SST, GSS or HTGS sequences; output can be restricted to showingonly the top ten matches.

In a PKD1-specific primer of the invention, the 5′ region contains atleast about ten contiguous nucleotides, generally at least about 12nucleotides, and usually about 14 to 18 nucleotides. In addition, the 3′region of the primer contains at least one 3′ terminal nucleotide, andcan include a sequence of at least about 2 to 6 nucleotides,particularly about 2 to 4 nucleotides. Where the 3′ region consists of asingle 3′ terminal nucleotide, the primer is selected such that the 3′terminal nucleotide is identical to a nucleotide that is 5′ and adjacentto the nucleotide sequence of the PKD1 gene to which the 5′ region ofthe primer can hybridize, and is different from a nucleotide that is 5′and adjacent to a nucleotide sequence of the PKD1 homolog to which the5′ region of the primer can hybridize, i.e., provides a mismatchednucleotide. Where the 3′ region of the PKD1-specific primer contains twoor more nucleotides, one or more of the nucleotides can be mismatched,and the mismatched nucleotide can, but need not include the 3′ terminalnucleotide, provided that when the mismatched nucleotide or nucleotidesdo not include the 3′ terminal nucleotide, the primer cannot be extendedwhen hybridized to a PKD1 gene homolog.

PKD1-specific primers of the invention are exemplified by primers thatcan selectively hybridize to a nucleotide sequence that flanks and iswithin about fifty nucleotides of a nucleotide sequence of SEQ ID NO:1selected from about nucleotides 2043 to 4209; nucleotides 17907 to22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615;nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides37329 to 41258; and nucleotides 41508 to 47320. A primer of theinvention is exemplified by any of SEQ ID NOS: 3 to 10, 12 to 17, 19 to51, and 61 to 113, and can have a sequence substantially identical toany of SEQ ID NOS:3 to 51 and 61 to 113, provided the sequence meets therequirements of a PKD1-specific primer as disclosed herein, and providedthe sequence is not a sequence as set forth in any of SEQ ID NO:11, SEQID NO:18, SEQ ID NO:52, and SEQ ID NO:60.

A primer is considered to be “substantially identical” to any of SEQ IDNOS:3 to 51 and 61 to 113 if the primer has at least about 80% or 85%,generally at least about 90%, usually at least about 95%, andparticularly at least about 99% sequence identity with one of SEQ IDNOS:3 to 51 and 61 to 113, and has a 5′ region and adjacentPKD1-specific 3′ region, wherein the 5′ region has a nucleotide sequencethat can hybridize to a PKD1 gene sequence or to a PKD1 gene sequenceand a PKD1 gene homolog sequence, and the 3′ region has a nucleotidesequence that selectively hybridizes only to a PKD1 gene sequence, andparticularly not to a PKD1 gene homolog sequence, as defined herein,except that a primer of the invention does not have a sequence as setforth in SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. Assuch, a primer of the invention can include one or a few, but no morethan about four or five, more or fewer nucleotide than a primer as setforth in SEQ ID NOS:3 to 51 and 61 to 113, provided the primer meets thefunctional requirements as defined herein.

The present invention also provides primer pairs. In one embodiment, aprimer pair of the invention comprising a forward and reversePKD1-specific primer as disclosed herein. As such, a primer pair of theinvention can amplify a portion of SEQ ID NO:1 including aboutnucleotides 2043 to 4209; nucleotides 17907 to 22489; nucleotides 22218to 26363; nucleotides 26246 to 30615; nucleotides 30606 to 33957;nucleotides 36819 to 37140; nucleotides 37329 to 41258; nucleotides41508 to 47320; or a combination thereof. In general, a primer pair ofthe invention can produce an amplification product of about tenkilobases or shorter, generally about 7500 bases or shorter, andparticularly about six kilobases or shorter. Primer pairs of theinvention are exemplified by a forward primer and a reverse primerselected from SEQ ID NOS:3 to 18, for example, by any of SEQ ID NOS:3and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ IDNOS:17 and 18; and SEQ ID NOS:9 and 113, which can be used to producePKD1-specific amplification products of about 0.3 kilobases to about 5.8kilobases.

As disclosed herein, a set of eight polymerase chain reaction (PCR)primer pairs can be used to prepare PKD1-specific amplification productsthat encompass all of the exons and their flanking introns within thereplicated region of the PKD1 gene. In view of the disclosed nucleotidesequences of the primers and of SEQ ID NO:1, it will be recognized thatadditional PCR primer pairs useful for a preparing PKD1-specific firstamplification product can be based on the exemplified primers and primerpairs, but can include one or few additional nucleotides (based on SEQID NO:1) at one or both ends of the exemplified primers, or can have oneor a few nucleotides of an exemplified primer deleted, and theirusefulness can be determined by comparing an amplification productgenerated using the derived or modified primer with a PKD1-specificamplification product as disclosed herein. As such, a primer pair based,for example, on SEQ ID NOS: 3 and 4 can be used to generate a PKD-1specific amplification product containing about nucleotides 2043 to 4209of SEQ ID NO:2, where in reference to “about” nucleotides 2043 to 4209of SEQ ID NO:2 accounts for the disclosure that a primer pair used foramplification can be identical or substantially identical to SEQ ID NOS:3 and 4.

Accordingly, the present invention provides primer pairs comprising aforward primer and a reverse primer having nucleotide sequences as setforth in SEQ ID NOS:3 to 18; primer pairs exemplified by SEQ ID NOS:3and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ IDNOS:17 and 18; and SEQ ID NOS:9 and 113; and substantially identicalprimer pairs that comprise primers based on or derived from theexemplified primers, such primer pairs being useful for preparing aPKD1-specific amplification product. The primer pairs shown as SEQ IDNOS: 9 and 10 and SEQ ID NOS: 11 and 12 have been described byPhakdeekitcharoen et al. (supra, 2000), as have the PKD1 specificamplification products generated using these primers.

It should be recognized that certain primers and certain primer pairsexemplified herein are not considered to be encompassed within thepresent invention. For example, the primer set forth in SEQ ID NO:11 hasbeen described in U.S. Pat. No. 6,017,717 (which is incorporated hereinby reference; column 24, SEQ ID NO:15); and the primer set forth in SEQID NO:18 has been described by Watnick et al. (Hum. Mol. Genet.6:1473-1481, 1997, which is incorporated herein by reference; see page1479; KG8R25), and, therefore, neither of these primers is considered tobe a primer of the invention. Nevertheless, the primers set forth as SEQID NOS: 11 and 18 can be encompassed within the primer pairs of theinvention, including within various disclosed and exemplified primerpairs, for example, the primer pairs set forth as SEQ ID NOS:11 and 12and as SEQ ID NOS:17 and 18, as well as within combinations of two ormore primer pairs, for example, a combination comprising SEQ ID NOS:11and 12 and SEQ ID NOS:13 and 14.

The primers set forth in SEQ ID NO:9 and SEQ ID NO:10 have beendescribed by Watnick et al. (Am. J. Hum. Genet. 65:1561-1571, 1999,which is incorporated herein by reference) and, therefore, can bespecifically excluded from certain embodiments of the invention, asdesired, for example, as encompassed within the primers of theinvention. It should be recognized, however, that the combination of SEQID NOS:9 and 10 as a primer pair is not described by Watnick et al.(supra, 1999). SEQ ID NOS:49 to 51 and 61 to 105 also have beendescribed by Watnick et al. (supra, 1999) and, therefore, can bespecifically excluded from certain embodiments of the invention, asdesired.

Except as provided herein, a primer of the invention is exemplified byany of SEQ ID NOS:3 to 51 and 61 to 113, as well as substantiallyidentical oligonucleotide primers that are based on or derived from SEQID NOS:3 to 51 and 61 to 113. It should be recognized, however, that theprimer set forth as SEQ ID NO:12 is substantially similar to the primerdesignated TWR2 by Watnick et al. (Mol. Cell 2:247-251, 1998, which isincorporated herein by reference; page 250;5′-GCAGGGTGAGCAGGTGGGGCCATCCTA-3′; SEQ ID NO:60), and that the primerset forth as SEQ ID NO:10 is substantially identical to SEQ ID NO:13 inU.S. Pat. No. 6,071,717 (5′-AGGTCAACGTGGGCCTCCAAGTAGT-3; SEQ ID NO:52).As such, a primer having the nucleotide sequence of SEQ ID NO:52 or ofSEQ ID NO:60 is specifically excluded from the primers that otherwisewould be encompassed within the scope of primers that have a sequencesubstantially identical to the sequence of the primer set forth as SEQID NO:12 or SEQ ID NO:10, respectively.

The present invention also provides an isolated mutant PKD1polynucleotide, or an oligonucleotide portion thereof comprising amutation as disclosed herein. As used herein, the term “isolated” or“purified,” when used in reference to a polynucleotide, oligonucleotide,or polypeptide, means that the material is in a form other than that inwhich it normally is found in nature. Thus, where a polynucleotide orpolypeptide occurs in a cell in nature, an isolated polynucleotide orpurified polypeptide can be one that separated, at least in part, fromthe materials with which it is normally associated. In general, anisolated polynucleotide or a purified polypeptide is present in a formin which it constitutes at least about 5 to 10% of a composition,usually 20% to 50% of a composition, particularly about 50% to 75% of acomposition, and preferably about 90% to 95% or more of a composition.Methods for isolating a polynucleotide or polypeptide are well known androutine in the art.

As part of or following isolation, a polynucleotide can be joined toother polynucleotides, such as DNA molecules, for example, formutagenesis studies, to form fusion proteins, or for propagation orexpression of the polynucleotide in a host. The isolatedpolynucleotides, alone or joined to other polynucleotides, such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Such polynucleotides, when introduced into host cells inculture or in whole organisms, nevertheless are considered “isolated”because they are not in a form in which they exist in nature. Similarly,the polynucleotides, oligonucleotides, and polypeptides can be presentin a composition such as a media formulation (solutions for introductionof polynucleotides, oligonucleotides, or polypeptides, for example, intocells or compositions or solutions for chemical or enzymatic reactionswhich are not naturally occurring compositions) and, therein remainisolated polynucleotides, oligonucleotides, or polypeptides within themeaning of that term as it is employed herein. An isolatedpolynucleotide can be a polynucleotide that is not immediatelycontiguous with nucleotide sequences with which it is immediatelycontiguous in a genome or other naturally occurring cellular DNAmolecule in nature. Thus, a recombinant polynucleotide, which cancomprise a polynucleotide incorporated into a vector, an autonomouslyreplicating plasmid, or a virus; or into the genomic DNA of a prokaryoteor eukaryote, which does not normally express a PKD1 polypeptide.

As used herein, the term “polynucleotide” or “oligonucleotide” or“nucleotide sequence” or the like refers to a polymer of two or morenucleotides or nucleotide analogs. The polynucleotide can be aribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, and canbe single stranded or double stranded DNA or RNA, or a double strandedDNA:RNA hybrid. A polynucleotide or oligonucleotide can contain one ormore modified bases, for example, inosine or a tritylated base. Thebonds linking the nucleotides in a polymer generally are phosphodiesterbonds, but can be other bonds routinely used to link nucleotidesincluding, for example, phosphorothioate bonds, thioester bonds, and thelike. A polynucleotide also can be a chemically, enzymatically ormetabolically modified form.

As used herein, the term “mutant PKD1 polynucleotide” means a nucleotidesequence that has one or a few nucleotide changes as compared to thenucleotide sequence set forth as SEQ ID NO:1. The nucleotide change canbe a deletion, insertion or substitution, and can be silent such thatthere is no change in the reading frame of a polypeptide encoded by thePKD1 polynucleotide, or can be a change that results in an amino acidchange or in the introduction of a STOP codon into the polynucleotide,or a change in a nucleotide sequence involved in transcription ortranslation of the PKD1 polynucleotide, for example, a change thatresults in altered splicing of a PKD1 gene transcript into an mRNA (seeExample 2). As disclosed herein, a mutant PKD1 polynucleotide can be apolymorphic variant, which, other than one or a few nucleotide changeswith respect to SEQ ID NO:1, encodes a PKD1 polypeptide and does notcorrelate with a PKD1 associated disorder, particularly ADPKD, or can bea mutant PKD1 polynucleotide that contains one or more mutations thatcorrelate with a PKD1 associated disorder such as ADPKD (see Example 2).

For convenience of discussion and for use as a frame of reference, thePKD1 nucleotide sequence set forth in SEQ ID NO:1 is referred to as a“wild type PKD1 polynucleotide” or a “wild type PKD1 gene” sequence,and, similarly, the polypeptide set forth as SEQ ID NO:2 is referred toas a “wild type PKD1 polypeptide.” However, while the presence of thewild type PKD1 gene sequence (i.e., SEQ ID NO:1) in an individualcorrelates to the absence of ADPKD in the individual, it should berecognized that polymorphic variants of SEQ ID NO:1 also are found inindividuals that do not exhibit ADPKD or other PKD1-associated disorder.The term “variants” or “polymorphic variants” is used herein to refer tomutant PKD1 polynucleotide sequences (with respect to SEQ ID NO:1) thatdo not correlate with the signs or symptoms characteristic of a PKD1associated disorder such as ADPKD. Variant PKD1 polynucleotides include,for example, nucleotide substitutions that do not result in a change inthe encoded amino acid, i.e., silent mutations, such as G4885A, in whichthe wild type and mutant codons both encode a threonine (T1558T), andC6058T, in which the wild type and mutant codons both, encode a serine(S1949S; see Example 2; see, also, Phakdeekitcharoen et al., supra,2000); those that do not segregate with the disease, or those that arefound in a panel of unaffected individuals. As such, it should berecognized that the term “mutant PKD1 polynucleotide” broadlyencompasses PKD1 variants, which do not correlate with a PKD1 associateddisorder, as well as mutant PKD1 polynucleotides that correlate or areassociated with a PKD1 associated disorder.

Examples of mutant PKD1 polynucleotide sequences, including variant PKD1polynucleotide sequence, include sequences substantially as set forth inSEQ ID NO:1, but having a mutation at nucleotide 474, wherein nucleotide474 is a T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide3110, wherein nucleotide 3110 is a C; a position corresponding tonucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide 3707,wherein nucleotide 3707 is an A; nucleotide 4168, wherein nucleotide4168 is a T; nucleotide 4885, wherein nucleotide 4885 is an A;nucleotide 5168, wherein nucleotide 5168 is a T; nucleotide 6058,wherein nucleotide 6058 is a T; nucleotide 6078, wherein nucleotide 6078is an A; nucleotide 6089, wherein nucleotide 6089 is a T; nucleotide6195, wherein nucleotide 6195 is an A; nucleotide 6326, whereinnucleotide 6326 is a T; a position corresponding to nucleotides 7205 to7211, wherein nucleotides 7205 to 7211 are deleted; nucleotide 7376,wherein nucleotide 7376 is a C; a nucleotide sequence corresponding tonucleotides 7535 to 7536, wherein a GCG nucleotide sequence is insertedbetween nucleotides 7535 and 7536; nucleotide 7415, wherein nucleotide7415 is a T; nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide7696, wherein nucleotide 7696 is a T; nucleotide 7883, whereinnucleotide 7883 is a T; nucleotide 8021, wherein nucleotide 8021 is anA; a nucleotide sequence corresponding to nucleotide 8159 to 8160,wherein nucleotides 8159 to 8160 are deleted; nucleotide 8298, whereinnucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G;nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326,wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367is a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide10143, wherein nucleotide 10143 is a G; nucleotide 10234, whereinnucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide 10255is a T; or a combination thereof (see Example 2; see, also, Tables 3 and4). Examples of a mutant PKD1 polynucleotide of the invention alsoinclude a polynucleotide that encodes a PKD1 polypeptide havingsubstantially as set forth in SEQ ID NO:2, but having an A88V, W967R,G1166S; V1956E; R1995H; R2408C; D2604N; L2696R, R2985G, R3039C, V3285I,H3311R mutation, or a combination thereof, as well as polypeptides thathave, for example, an addition of a Gly residue between amino acidresidues 2441 and 2442 of SEQ ID NO:2 due to an insertion, or thatterminate with amino acid 3000 of SEQ ID NO:2 due to the presence of aSTOP codon at the position in SEQ ID NO:1 that would otherwise encodeamino acid 3001 (see, also, Table 4; Example 2).

Additional examples of mutant PKD1 polynucleotides of the inventioninclude polynucleotide sequences that selectively hybridize to thecomplements of the polynucleotide sequences, or oligonucleotide portionsthereof, as disclosed herein, under highly stringent hybridizationconditions, e.g., hybridization to filter-bound DNA in 0.5M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., Current Protocols inMolecular Biology, (Green Publishing Associates, Inc., and John Wiley &Sons, Inc., New York 1989), and supplements; see p. 2.10.3; Sambrook etal., Molecular Cloning: A laboratory manual (Cold Spring HarborLaboratory Press, 1989), which are incorporated herein by reference), aswell as polynucleotides that encode a PKD1 polypeptide substantially asset forth in SEQ ID NO:2, but having one or more mutations; or an RNAcorresponding to such a polynucleotide.

A polynucleotide or polypeptide sequence that is “substantiallyidentical” to a PKD1 polynucleotide of SEQ ID NO:1 or a polypeptidesequence of SEQ ID NO:2 generally is at least 80% or 85%, usually atleast about 90%, and particularly at least about 95%, and preferably atleast about 99% identical to the nucleotide sequence or amino acidsequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, respectively. Itshould be recognized, however, that a mutation in a PKD1 gene sequencecan result in the expression of a truncated PKD1 polypeptide, or even acomplete loss of expression of the PKD1 polypeptide. As such, while amutant PKD1 polynucleotide is identified as being substantiallyidentical to SEQ ID NO:1, it may not always be possible to make the samecomparison with respect to the encoded polypeptides. In one aspect ofthe invention, a polynucleotide or polypeptide sequence that issubstantially identical to SEQ ID NO:1 or 2 will vary at one or moresites having a mutation, for example, a mutation present in a mutantPKD1 polynucleotide as set forth in the preceding paragraph. Sequenceidentity can be measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison Wis. 53705).

A polynucleotide or oligonucleotide portion thereof of the invention canbe useful, for example, as a probe or as a primer for an amplificationreaction. Reference to an “oligonucleotide portion” of a mutant PKD1polynucleotide means a nucleotide sequence of the mutant PKD1polynucleotide that is less than the full length polynucleotide.Generally, a polynucleotide useful as a probe or a primer contains atleast about 10 nucleotides, and usually contains about 15 to 30nucleotides or more (see, for example, Tables 1 and 2). Polynucleotidescan be prepared by any suitable method, including, for example, byrestriction enzyme digestion of an appropriate polynucleotide, by directchemical synthesis using a method such as the phosphotriester method(Narang et al., 1979, Meth. Enzymol., 68:90-99); the phosphodiestermethod (Brown et al., 1979, Meth. Enzymol., 68:109-151); thediethylphosphoramidite method (Beaucage et al., 1981, Tetrahedron Lett.,22:1859-1862); the triester method (Matteucci et al., 1981, J. Am. Chem.Soc., 103:3185-3191), including by automated synthesis methods; or by asolid support method (see, for example, U.S. Pat. No. 4,458,066). Inaddition, a polynucleotide or oligonucleotide can be prepared usingrecombinant DNA methods as disclosed herein or otherwise known in theart.

An oligonucleotide of the invention can include a portion of a mutantPKD1 polynucleotide, including, for example, a sequence substantiallyidentical to that of SEQ ID NO:1, except wherein nucleotide 474 is a T;or wherein nucleotide 487 is an A; or wherein nucleotide 3110 is a C; orwherein nucleotide 8298 is a G; or wherein nucleotide 9164 is a G; orwherein nucleotide 9213 is an A; or wherein nucleotide 9326 is a T; orwherein nucleotide 9367 is a T; or wherein nucleotide 10064 is an A; orwherein nucleotide 10143 is a G; or wherein nucleotide 10234 is a C; orwherein nucleotide 10255 is a T; or wherein the oligonucleotide containsa combination of such substitutions with respect to SEQ ID NO:1. Thus,as disclosed herein, the oligonucleotide can be any length and canencompass one or more of the above mutations.

An oligonucleotide of the invention can selectively hybridize to amutant PKD1 polynucleotide sequence as disclosed herein. As such, theoligonucleotide does not hybridize substantially, if at all, to a wildtype PKD1 polynucleotide (i.e., to SEQ ID NO:1). As used herein, theterm “selectively hybridize” refers to the ability of an oligonucleotide(or polynucleotide) probe to hybridize to a selected sequence, but notto a highly related nucleotide sequence. For example, a oligonucleotideof the invention selectively hybridizes to a mutant PKD1 polynucleotide,but not substantially to a corresponding sequence of SEQ ID NO:1. Assuch, hybridization of the oligonucleotide to SEQ ID NO:1 generally isnot above background, or, if some hybridization occurs, is at leastabout ten-fold less than the amount of hybridization that occurs withrespect to the mutant PKD1 polynucleotide.

In addition, the term “hybridize” is used herein to have its commonlyunderstood meaning of two nucleotide sequences that can associate due toshared complementarity. As disclosed herein, a primer of the inventioncan hybridize to PDK1 gene and may also hybridize to a PDK1 genehomolog, but generally does not substantially hybridize to a nucleotidesequence other than a PKD1 gene or PKD1 gene homolog. Desiredhybridization conditions, including those that allow for selectivehybridization, can be obtained by varying the stringency of thehybridization conditions, based, in part, on the length of the sequencesinvolved, the relative G:C content, the salt concentration, and the like(see Sambrook et al., supra, 1989). Hybridization conditions that arehighly stringent conditions are used for selective hybridization and canbe used for hybridization of a primer or primer pair of the invention toa PKD1 gene or PKD1 gene homolog, and include, for example, washing in6×SSC/0.05% sodium pyrophosphate at about 37° C. (for 14 nucleotide DNAprobe), about 48° C. (for 17 nucleotide probe), about 55° C. (for a 20nucleotide probe), and about 60° C. (for a 23 nucleotide probe). Asdisclosed herein, polynucleotides that selectively hybridize to a mutantPKD1 polynucleotide provide a means to distinguish the mutant PKD1polynucleotide from a wild type PKD1 polynucleotide.

A polynucleotide or oligonucleotide of the invention can be used as aprobe to screen for a particular PKD1 variant or mutant of interest. Inaddition, the oligonucleotides of the invention include a PKD1 antisensemolecule, which can be useful, for example, in PKD1 polynucleotideregulation and amplification reactions of PKD1 polynucleotide sequences,including mutant PKD1 polynucleotide sequences. Further, sucholigonucleotides can be used as part of ribozyme or triple helixsequence for PKD1 gene regulation. Still further, such oligonucleotidescan be used as a component of diagnostic method, whereby the level ofPKD1 transcript can be determined or the presence of an ADPKD-causingallele can be detected. Further, such oligonucleotides can be used, forexample, to screen for and identify PKD1 homologs from other species.

The term “primer” or “PCR primer” refers to an isolated natural orsynthetic oligonucleotide that can act as a point of initiation of DNAsynthesis when placed under conditions suitable for primer extension.Synthesis of a primer extension product is initiated in the presence ofnucleoside triphosphates and a polymerase in an appropriate buffer at asuitable temperature. A primer can comprise a plurality of primers, forexample, where there is some ambiguity in the information regarding oneor both ends of the target region to be synthesized. For instance, if anucleic acid sequence is determined from a protein sequence, a primergenerated to synthesize nucleic acid sequence encoding the proteinsequence can comprise a collection of primers that contains sequencesrepresenting all possible codon variations based on the degeneracy ofthe genetic code. One or more of the primers in this collection will behomologous with the end of the target sequence or a sequence flanking atarget sequence. Likewise, if a conserved region shows significantlevels of polymorphism in a population, mixtures of primers can beprepared that will amplify adjacent sequences.

During PCR amplification, primer pairs flanking a target sequence ofinterest are used to amplify the target sequence. A primer pairtypically comprises a forward primer, which hybridizes to the 5′ end ofthe target sequence, and a reverse primer, which hybridizes to the 3′end of the target sequence. Except as otherwise provided herein, primersof the present invention are exemplified by those having the sequencesset forth as SEQ ID NOS:3 to 51 and 61 to 113 (see Tables 1 and 2).Forward primers are exemplified by SEQ ID NOS:3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49;and reverse primers are exemplified by SEQ ID NOS:4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and50. A primer pair of the invention includes at least one forward primerand at least one reverse primer that allows for generation of anamplification product, which can be a long range PKD1-specificamplification product or a nested amplification product of such anamplification product, including a forward and reverse primer as setforth in SEQ ID NOS:3 to 18 and of SEQ ID NOS:19 to 51 and 61 to 113,provided that the forward primer is 5′ (or upstream) of the reverseprimer with reference to a target polynucleotide sequence, and that theprimers are in sufficient proximity such that an amplification productcan be generated.

Nucleic acid sequences that encode a fusion protein can be produced andcan be operatively linked to expression control sequences. Such fusionproteins and compositions are useful in the development of antibodies orto generate and purify peptides and polypeptides of interest. As usedherein, the term “operatively linked” refers to a juxtaposition, whereinthe components so described are in a relationship permitting them tofunction in their intended manner. For example, an expression controlsequence operatively linked to a coding sequence is ligated such thatexpression of the coding sequence is achieved under conditionscompatible with the expression control sequences, whereas twooperatively linked coding sequences can be ligated such that they are inthe same reading frame and, therefore, encode a fusion protein.

As used herein, the term “expression control sequences” refers tonucleic acid sequences that regulate the expression of a nucleic acidsequence to which it is operatively linked. Expression control sequencesare operatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus, expressioncontrol sequences can include appropriate promoters, enhancers,transcription terminators, a start codon ATG) in front of aprotein-encoding gene, splicing signals for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of themRNA, and STOP codons. Control sequences include, at a minimum,components whose presence can influence expression, and can also includeadditional components whose presence is advantageous, for example,leader sequences and fusion partner sequences. Expression controlsequences can include a promoter.

A polynucleotide of the invention can comprise a portion of arecombinant nucleic acid molecule, which, for example, can encode afusion protein. The polynucleotide, or recombinant nucleic acidmolecule, can be inserted into a vector, which can be an expressionvector, and can be derived from a plasmid, a virus or the like. Theexpression vector generally contains an origin of replication, apromoter, and one or more genes that allow phenotypic selection oftransformed cells containing the vector. Vectors suitable for use in thepresent invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988);baculovirus-derived vectors for expression in insect cells; and thelike.

The choice of a vector will also depend on the size of thepolynucleotide sequence and the host cell to be employed in the methodsof the invention. Thus, the vector used in the invention can beplasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses,parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and thelike), or selected portions thereof (e.g., coat protein, spikeglycoprotein, capsid protein). For example, cosmids and phagemids aretypically used where the specific nucleic acid sequence to be analyzedor modified is large because these vectors are able to stably propagatelarge polynucleotides. Cosmids and phagemids are particularly suited forthe expression or manipulation of the PKD1 polynucleotide of SEQ ID NO:1or a mutant PKD1 polynucleotide.

In yeast, a number of vectors containing constitutive or induciblepromoters can be used (see Ausubel et al., supra, 1989; Grant et al.,Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning, Vol. II, IRLPress, Washington D.C., Ch. 3, 1986; and Bitter, Meth. Enzymol.152:673-684, 1987; and The Molecular Biology of the Yeast Saccharomyces,Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982).A constitutive yeast promoter such as ADH or LEU2 or an induciblepromoter such as GAL can be used (“Cloning in Yeast,” Ch. 3, Rothstein,In “DNA Cloning” Vol. 11, A Practical ively hybridize to SEQ ID NO:1 andamplify portions of SEQ ID NO:1 comprising about nucleotides 2043 to4290; nuclNA sequences into the yeast chromosome. The construction ofexpression vectors and the expression of genes in transfected cellsinvolves the use of molecular cloning techniques also well known in theart (see Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination.

A polynucleotide or oligonucleotide can be contained in a vector and canbe introduced into a cell by transformation or transfection of the cell.By “transformation” or “transfection” is meant a permanent (stable) ortransient genetic change induced in a cell following incorporation ofnew DNA (i.e., DNA exogenous to the cell). Where the cell is a mammaliancell, a permanent genetic change is generally achieved by introductionof the DNA into the genome of the cell.

A transformed cell or host cell can be any prokaryotic or eukaryoticcell into which (or into an ancestor of which) has been introduced, bymeans of recombinant DNA techniques, a polynucleotide sequence of theinvention or fragment thereof. Transformation of a host cell can becarried out by conventional techniques as are well known to thoseskilled in the art. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art, or using MgCl₂or RbCl. Transformation can also be performed after forming a protoplastof the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection include theuse of calcium phosphate co-precipitates, conventional mechanicalprocedures such as microinjection, electroporation, insertion of aplasmid encased in liposomes, or the use of virus vectors, or othermethods known in the art. One method uses a eukaryotic viral vector,such as simian virus 40 (SV40) or bovine papillomavirus, to transientlyinfect or transform eukaryotic cells and express the protein.(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed.,1982). Preferably, a eukaryotic host is utilized as the host cell asdescribed herein. The eukaryotic cell can be a yeast cell (e.g.,Saccharomyces cerevisiae), or can be a mammalian cell, including a humancell.

A variety of host-expression vector systems can be utilized to express aPKD1 polynucleotide sequence such as SEQ ID NO:1, a coding sequence ofSEQ ID NO:1 or a mutant PKD1 polynucleotide. Such host-expressionsystems represent vehicles by which the nucleotide sequences of interestcan be produced and subsequently purified, and also represent cellsthat, when transformed or transfected with the appropriate nucleotidecoding sequences, can express a PKD1 protein, including a PKD1 variantor mutant polypeptide or peptide portion thereof in situ. Such cellsinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing a PKD1polynucleotide, or oligonucleotide portion thereof (wild type, variantor other mutant); yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing a PKD1 polynucleotide,or oligonucleotide portions thereof (wild type, variant or other PKD1mutant); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing a PKD1 polynucleotide, oroligonucleotide portion thereof (wild type, PKD1 variant or othermutant); plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing a mutant PKD1 polynucleotide, or oligonucleotideportion thereof; or mammalian cell systems (e.g., COS, CHO, BHK, 293,3T3) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the PKD1protein (wild type, variant or other PKD1 mutant) being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies, which can be used to identify or diagnosePKD1-associated diseases or disorders, or to screen peptide libraries,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified can be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a PKD1 polynucleotide,or oligonucleotide portion thereof (wild type, variant or other mutant)can be ligated individually into the vector in frame with the lac Zcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, Nucl. Acids Res. 13:3101-3109, 1985; Van Heeke and Schuster,J. Biol. Chem. 264:5503-5509, 1989); and the like. pGEX vectors can alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned PKD1 protein, variant ormutant can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. A PKD1 polynucleotide, or oligonucleotideportion thereof can be cloned individually into non-essential regions(for example the polyhedrin gene) of the virus and placed under controlof an AcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of a PKD1 polynucleotide, or oligonucleotide portion thereofwill result in inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (see Smith et al., 1983, J. Virol. 46:584; U.S. Pat.No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, a PKD1 polynucleotide, or oligonucleotide portion thereof, canbe ligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene can then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome such as the E1 or E3 region results in a recombinant virus thatis viable and capable of expressing a PKD1 protein (e.g., wild-type,variants or mutants thereof) in infected hosts (Logan and Shenk, Proc.Natl. Acad. Sci., USA 81:3655-3659, 1984). Specific initiation signalscan also be required for efficient translation of an inserted PKD1sequence. These signals include the ATG initiation codon and adjacentsequences. Where an entire PKD1 polynucleotide, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals can be needed. However, where only a portion of a PKD1 sequenceis inserted, exogenous translational control signals, including, forexample, an ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, and the like (see Bittneret al., Meth. Enzymol. 153:516-544, 1987).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes theexpressed polypeptide in a specific fashion. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein being expressed. To this end, eukaryotic hostcells which possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of thepolypeptide can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and thelike.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express aPKD1 protein, including wild-type, variants or mutants of PKD1, can beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoterand/or enhancer sequences, transcription terminators, polyadenylationsites, and the like), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be grown for 1-2days in an enriched media, then switched to selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci, which can be cloned and expanded intocell lines. This method can advantageously be used to engineer celllines that express a PKD1 variant or mutant polypeptide. Such engineeredcell lines can be particularly useful in screening and evaluation ofcompounds that affect the endogenous activity of a variant or mutantPKD1 polypeptide. Such engineered cell lines also can be useful todiscriminate between factors that have specific vs. non-specificeffects. In particular, mutant cell lines should lack key functions, andvarious mutations can be used to identify key functional domains usingin vivo assays.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprf⁻ or aprf⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., Proc.Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad.Sci. USA 78:1527, 1981); gpt, which confers resistance to mycophenolicacid Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., J. Mol. Biol. 150:1, 1981); and hygro, which confers resistanceto hygromycin (Santerre et al., Gene 30:147, 1984) genes. Accordingly,the invention provides a vector that contains a mutant PKD1polynucleotide, or oligonucleotide portion thereof, or one or moreprimers or their complements, including an expression vector thatcontains any of the foregoing sequences operatively associated with aregulatory element that directs the expression of a coding sequence orprimer; and also provides a host cell that contains any of the foregoingsequences, alone or operatively associated with a regulatory element,which can directs expression of a polypeptide encoded thepolynucleotide, as appropriate.

In addition to mutant PKD1 polynucleotide sequences disclosed herein,homologs of mutant PKD1 polynucleotide of the invention, including anon-human species, can be identified and isolated by molecularbiological techniques well known in the art. Further, mutant PKD1alleles and additional normal alleles of the human PKD1 polynucleotide,can be identified using the methods of the invention. Still further,there can exist genes at other genetic loci within the human genome thatencode proteins having extensive homology to one or more domains of thePKD1 polypeptide (SEQ ID NO:2). Such genes can also be identifiedincluding associated variants and mutants by the methods of theinvention.

A homolog of a mutant PKD1 polynucleotide sequence can be isolated byperforming a polymerase chain reaction (PCR; see U.S. Pat. No.4,683,202, which is incorporated herein by reference) using twooligonucleotide primers, which can be selected, for example, from amongSEQ ID NOS:3 to 51, preferably from among SEQ ID NOS: 3 to 18, or can bedegenerate primer pools designed on the basis of the amino acidsequences of a PKD1 polypeptide such as that set forth in SEQ ID NO:2 ora mutant thereof as disclosed herein. The template for the reaction canbe cDNA obtained by reverse transcription of mRNA prepared from human ornon-human cell lines or tissue known to express a PKD1 allele or PKD1homologue. The PCR product can be subcloned and sequenced or manipulatedin any number of ways (e.g., further manipulated by nested PCR) toinsure that the amplified sequences represent the sequences of a PKD1 ora PKD mutant polynucleotide sequence. The PCR fragment can then be usedto isolate a full length PKD1 cDNA clone (including clones containing amutant PKD1 polynucleotide sequence) by labeling the amplified fragmentand screening a nucleic acid library (e.g., a bacteriophage cDNAlibrary). Alternatively, the labeled fragment can be used to screen agenomic library (for review of cloning strategies, see, for example,Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).

The present invention also provides a purified mutant PKD1 polypeptide,or a peptide portion thereof. As disclosed herein, a mutant PKD1polypeptide has an amino acid sequence substantially identical to SEQ IDNO:2, and includes a mutation resulting in the deletion, addition(insertion), or substitution of an amino acid of SEQ ID NO:2, or istruncated with respect to SEQ ID NO:2. Examples of such mutationsinclude, with respect to SEQ ID NO:2, an A88V, W967R, G1166S; V1956E;R1995H; R2408C; D2604N; L2696R, R2985G, R3039C, V3285I, or H3311Rmutation, an addition of a Gly residue between amino acid residues 2441and 2442 of SEQ ID NO:2 due to an insertion, or a truncated PKD1polypeptide terminates with amino acid 3000 of SEQ ID NO:2 due to thepresence of a STOP codon at the position in SEQ ID NO:1 that wouldotherwise encode amino acid 3001; as well as mutant PKD1 polypeptideshaving a combination of such mutations (see Table 4).

A mutant PKD1 polypeptide or peptide portion thereof can contain one ormore of the exemplified mutations. As used herein, reference to apeptide portion of SEQ ID NO:2 or of a mutant PKD1 polypeptide refers toa contiguous amino acid sequence of SEQ ID NO:2 or of SEQ ID NO:2including a mutation as disclosed herein, respectively, that containsfewer amino acids than full length wild type PKD1 polypeptide.Generally, a peptide portion of a PKD1 polypeptide or a mutant PKD1polypeptide contains at least about five amino acids (or amino acidderivatives or modified amino acids), each linked by a peptide bond or amodified form thereof, usually contains at least about eight aminoacids, particularly contains about ten amino acids, and can containtwenty or thirty or more amino acids of SEQ ID NO:2. In particular,where the peptide is a peptide portion of a mutant PKD1 polypeptide, thepeptide includes a mutant amino acid with respect to SEQ ID NO:2.

The mutant PKD1 polypeptides and peptide fragments thereof of theinvention include a PKD1 polypeptide or peptide having a sequencesubstantially identical to that set forth in SEQ ID NO:2, and having oneor a combination of the following mutations: A88V, W967R, L2696R,R2985G, R3039C, V3285I, or H3311R, or a mutation resulting intermination of the mutant PKD1 polypeptide at amino acid 3000 (withrespect to SEQ ID NO:2) due to the presence of a STOP codon at theposition that otherwise would encode amino acid 3001. The wild type PKD1polypeptide (SEQ ID NO:2) contains 4303 amino acid residues and has apredicted molecular mass of approximately 467 kilodaltons (kDa). Furtherencompassed by the present invention are mutant PKD1 polypeptides thatare truncated with respect to SEQ ID NO:2, for example, a mutation ofSEQ ID NO:1 resulting in a G9213A, which results in prematuretermination of the encoded PKD1 polypeptide (see Example 2). Suchtruncated products can be associated with PKD1-associated disorders suchas ADPKD (see, also, Table 4).

PKD1 polypeptides that are functionally equivalent to a wild type PKD1polypeptide, including variant PKD1 polypeptides, which can contain adeletion, insertion or substitution of one or more amino acid residueswith respect to SEQ ID NO:2, but that nevertheless result in a phenotypethat is indistinguishable from that conferred by SEQ ID NO:2, areencompassed within the present invention. Such amino acid substitutions,for example, generally result in similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity, amphipatic nature or thelike of the residues involved. For example, negatively charged aminoacids include aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; amino acids with uncharged polar headgroups having similar hydrophilicity values include the following:leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine,serine, threonine, phenylalanine and tyrosine. In many cases, however, anucleotide substitution can be silent, resulting in no change in theencoded PKD1 polypeptide (see Example 2). Such variant PKD1polynucleotides are exemplified by those encoded by the variant PKD1polynucleotide sequences substantially identical to SEQ ID NO:1 (SEQ IDNO:2), but containing (encoding) G487A (A92A), C9367T (G3052G), T10234C(L3341L), and G10255T (R3348R) as shown in Table 3 (see, also, Example2), and by C9494T (L3095L).

Mutant PKD1 polypeptides and peptide portions thereof that aresubstantially identical to the PKD1 polypeptide SEQ ID NO:2 or peptideportions thereof, which cause ADPKD symptoms, are encompassed within thescope of the invention. Such mutant PKD1 polypeptides and peptideportions thereof can include dominant mutant PKD1 polypeptides, or PKD1related polypeptides functionally equivalent to such mutant PKD1polypeptides. Examples of mutant PKD1 polypeptide sequences include apolypeptide sequences substantially identical to SEQ ID NO:2 having oneor more amino acid substitutions such as A88V, W967R, L2696R, R2985G,R3039C, V3285I, or H3311R, or truncated after amino acid 3000. A peptideportion of a mutant PKD1 polypeptide can be 3, 6, 9, 12, 20, 50, 100 ormore amino acid residues in length, and includes at least one of themutations identified above.

A PKD1 wild type or mutant polypeptide, or peptide portions thereof, canbe purified from natural sources, as discussed below; can be chemicallysynthesized; or can be recombinantly expressed. For example, one skilledin the art can synthesize peptide fragments corresponding to a mutatedportion of the PKD1 polypeptide as set forth in SEQ ID NO:2 (e.g.,including residue 3110) and use the synthesized peptide fragment togenerate polyclonal and monoclonal antibodies. Synthetic polypeptides orpeptides can be prepared by chemical synthesis, for example, solid-phasechemical peptide synthesis methods, which are well known (see, forexample, Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963; Stewart andYoung, Solid Phase Peptide Synthesis, 2d ed., Pierce Chemical Co.,Rockford Ill., pages 11-12), and have been employed in commerciallyavailable laboratory peptide design and synthesis kits (CambridgeResearch Biochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of Geysen et al., Proc. Natl. Acad.Sci., USA, 81:3998 (1984) and provide for synthesizing peptides upon thetips of a multitude of rods or pins, each of which is connected to asingle plate. When such a system is utilized, a plate of rods or pins isinverted and inserted into a second plate of corresponding wells orreservoirs, which contain solutions for attaching or anchoring anappropriate amino acid to the tips of the pins or rods. By repeatingsuch a process step, i.e., inverting and inserting the tips of the rodsor pins into appropriate solutions, amino acids are built into desiredpeptides.

A number of available FMOC peptide synthesis systems are available. Forexample, assembly of a polypeptide or fragment can be carried out on asolid support using an Applied Biosystems, Inc., Model 431A automatedpeptide synthesizer. Such equipment provides ready access to thepeptides of the invention, either by direct synthesis or by synthesis ofa series of fragments that can be coupled using other known techniques.Accordingly, methods for the chemical synthesis of polypeptides andpeptides are well-known to those of ordinary skill in the art, e.g.,peptides can be synthesized by solid phase techniques, cleaved from theresin and purified by preparative high performance liquid chromatography(see, e.g., Creighton, 1983, Proteins: Structures and MolecularPrinciples, W. H. Freeman & Co., N.Y., pp. 50-60). The composition ofthe synthetic peptides can be confirmed by amino acid analysis orsequencing; e.g., using the Edman degradation procedure (see e.g.,Creighton, 1983, supra at pp. 34-49). Thus, fragments of the PKD1polypeptide, variant, or mutant can be chemically synthesized. Peptidescan then be used, for example, to generate antibodies useful in thedetection of PKD1 variants and mutants, as well as the diagnosis ofPKD1-associated disorder (e.g., ADPKD).

A PKD1 polypeptide or peptide, including variants or mutants of theinvention, can be substantially purified from natural sources (e.g.,purified from cells) using protein separation techniques, well known inthe art. Such methods can separate the PKD1 polypeptide away from atleast about 90% (on a weight basis), and from at least about 99% ofother proteins, glycoproteins, and other macromolecules normally foundin such natural sources. Such purification techniques can include, butare not limited to ammonium sulfate precipitation, molecular sievechromatography, and/or ion exchange chromatography. Alternatively, oradditionally, the PKD1 polypeptide, variant, or mutant can be purifiedby immunoaffinity chromatography using an immunoabsorbent column towhich an antibody is immobilized that is capable of specifically bindingthe PKD1 polypeptide, variant, or mutant. Such an antibody can bemonoclonal or polyclonal in origin. For example, an antibody thatspecifically binds to a mutant PKD1 polypeptide does not bind to awild-type PKD1 polypeptide or peptide thereof. If the PKD1 polypeptideis glycosylated, the glycosylation pattern can be utilized as part of apurification scheme via, for example, lectin chromatography.

The cellular sources from which the PKD1 polypeptide, variant, ormutants thereof can be purified include, for example, those cells thatare shown by northern and/or western blot analysis to express a PKD1polynucleotide, variant, or mutant sequence. Preferably, such cellularsources are renal cells including, for example, renal tubular epithelialcells, as well as biliary duct cells, skeletal muscle cells, lungalveolar epithelial cell, placental cells, fibroblasts, lymphoblasts,intestinal epithelial cells, and endothelial cells. Other sourcesinclude biological fluids, fractionated cells such as organellepreparations, or tissues obtained from a subject. Examples of biologicalfluids of use with the invention are blood, serum, plasma, urine,mucous, and saliva. Tissue or cell samples can also be used with theinvention. The samples can be obtained by many methods such as cellularaspiration, or by surgical removal of a biopsy sample.

PKD1 polypeptides, variants, or mutants of the invention can be secretedout of the cell. Such extracellular font's of the PKD1 polypeptide ormutants thereof can preferably be purified from whole tissue rather thancells, utilizing any of the techniques described above. PKD1 expressingcells such as those described above also can be grown in cell culture,under conditions well known to those of skill in the art. PKD1polypeptide or mutants thereof can then be purified from the cell mediausing any of the techniques discussed above.

A PKD1 polypeptide, variant, or mutant can additionally be produced byrecombinant DNA technology using the PKD1 nucleotide sequences, variantsand mutants described above coupled with techniques well known in theart. Alternatively, RNA capable of encoding PKD1 polypeptides, orpeptide fragments thereof, can be chemically synthesized using, forexample, automated or semi-automated synthesizers (see, for example,“Oligonucleotide Synthesis”, 1984, Gait, ed., IRL Press, Oxford, whichis incorporated herein by reference).

When used as a component in the assay systems described herein, themutant PKD1 polypeptide or peptide can be labeled, either directly orindirectly, to facilitate detection of a complex formed between the PKD1polypeptide and an antibody or nucleic acid sequence, for example. Anyof a variety of suitable labeling systems can be used including, but notlimited to, radioisotopes such as ¹²⁵I, enzyme labeling systems such asbiotin-avidin or horseradish peroxidase, which generates a detectablecolorimetric signal or light when exposed to substrate, and fluorescentlabels.

The present invention also provides antibodies that specifically bind aPKD1 mutant or PKD1 variant, except that, if desired, an antibody of theinvention can exclude an antibody as described in U.S. Pat. No.5,891,628, which is incorporated herein by reference, or an antibodythat that specifically binds a PKD1 mutant as described in U.S. Pat. No.5,891,628. Antibodies that specifically bind a mutant PKD1 polypeptideare useful as diagnostic or therapeutic reagents and, therefore, can beused, for example, in a diagnostic assay for identifying a subjecthaving or at risk of having ADPKD, and are particularly convenient whenprovided as a kit.

As used herein, the term “specifically binds,” when used in reference toan antibody and an antigen or epitopic portion thereof, means that theantibody and the antigen (or epitope) have a dissociation constant of atleast about 1×10⁻⁷, generally at least about 1×10⁻⁸, usually at leastabout 1×10⁻⁹, and particularly at least about 1×10⁻¹⁰ or less. Methodsfor identifying and selecting an antibody having a desired specificityare well known and routine in the art (see, for example, Harlow andLane, “Antibodies: A Laboratory Manual” (Cold Spring Harbor Pub. 1988),which is incorporated herein by reference.

Methods for producing antibodies that can specifically bind one or morePKD1 polypeptide epitopes, particularly epitopes unique to a mutant PKD1polypeptide, are disclosed herein or otherwise well known and routine inthe art. Such antibodies can be polyclonal antibodies or monoclonalantibodies (mAbs), and can be humanized or chimeric antibodies, singlechain antibodies, anti-idiotypic antibodies, and epitope-bindingfragments of any of the above, including, for example, Fab fragments,F(ab′)₂ fragments or fragments produced by a Fab expression library.Such antibodies can be used, for example, in the detection of PKD1polypeptides, or mutant PKD1 polypeptides, including variant PKD1polypeptides, which can be in a biological sample, or can be used forthe inhibition of abnormal PKD1 activity. Thus, the antibodies can beutilized as part of ADPKD treatment methods, as well as in diagnosticmethods, for example, to detect the presence or amount of a PKD1polypeptide.

For the production of antibodies that bind to PKD1, including a PKD1variant or PKD1 mutant, various host animals can be immunized byinjection with a PKD1 polypeptide, mutant polypeptide, variant, or aportion thereof. Such host animals can include but are not limited to,rabbits, mice, and rats. Various adjuvants can be used to increase theimmunological response, depending on the host species, including, butnot limited to, Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacillus Calmette-Guerin) or Corynebacterium parvum.

Antibodies that bind to a mutant PKD1 polypeptide, or peptide portionthereof, of the invention can be prepared using an intact polypeptide orfragments containing small peptides of interest as the immunizingantigen. The polypeptide or a peptide used to immunize an animal can bederived from translated cDNA or chemical synthesis, and can beconjugated to a carrier protein, if desired. Such commonly used carriersthat can be chemically coupled to the peptide include keyhole limpethemocyanin, thyroglobulin, bovine serum albumin, tetanus toxoid andothers as described above or otherwise known in the art. The coupledpolypeptide or peptide is then used to immunize the animal and antiserumcan be collected. If desired, polyclonal or monoclonal antibodies can bepurified, for example, by binding to and elution from a matrix to whichthe polypeptide or a peptide to which the antibodies were raised isbound. Any of various techniques commonly used in immunology forpurification and/or concentration of polyclonal antibodies, as well asmonoclonal antibodies, can be used (see for example, Coligan, et al.,Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, whichis incorporated herein by reference).

Anti-idiotype technology can be used to produce monoclonal antibodiesthat mimic an epitope. For example, an anti-idiotypic monoclonalantibody made to a first monoclonal antibody will have a binding domainin the hypervariable region that is the image of the epitope bound bythe first monoclonal antibody. Antibodies of the invention includepolyclonal antibodies, monoclonal antibodies, and fragments ofpolyclonal and monoclonal antibodies that specifically bind to a mutantPKD1 polypeptide or peptide portion thereof.

The preparation of polyclonal antibodies is well known to those skilledin the art (see, for example, Green et al., Production of PolyclonalAntisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (HumanaPress 1992); Coligan et al., Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology,section 2.4.1 (1992), which are incorporated herein by reference). Thepreparation of monoclonal antibodies likewise is conventional (see, forexample, Kohler and Milstein, Nature, 256:495, 1975, which isincorporated herein by reference; see, also Coligan et al., supra,sections 2.5.1-2.6.7; and Harlow et al., supra, 1988). Briefly,monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtain Blymphocytes, fusing the B lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (seeColigan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barneset al., Purification of Immunoglobulin G (IgG), in Methods in MolecularBiology, Vol. 10, pages 79-104 (Humana Press 1992)). Methods of in vitroand in vivo multiplication of hybridoma cells expressing monoclonalantibodies is well-known to those skilled in the art. Multiplication invitro can be carried out in suitable culture media such as Dulbecco'sModified Eagle Medium or RPMI 1640 medium, optionally replenished by amammalian serum such as fetal calf serum or trace elements andgrowth-sustaining supplements such as normal mouse peritoneal exudatecells, spleen cells, bone marrow macrophages. Production in vitroprovides relatively pure antibody preparations and allows scale-up toyield large amounts of the desired antibodies. Large scale hybridomacultivation can be carried out by homogenous suspension culture in anairlift reactor, in a continuous stirrer reactor, or in immobilized orentrapped cell culture. Multiplication in vivo can be carried out byinjecting cell clones into mammals histocompatible with the parentcells, e.g., syngeneic mice, to cause growth of antibody-producingtumors. Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane tetramethylpentadecane prior toinjection. After one to three weeks, the desired monoclonal antibody isrecovered from the body fluid of the animal.

Therapeutic applications for antibodies disclosed herein are also partof the present invention. For example, antibodies of the presentinvention can be derived from subhuman primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in Goldenberg et al., International ApplicationPublication No. WO 91/11465, 1991; Losman et al., Int. J. Cancer,46:310, 1990, which are incorporated herein by reference.

An anti-PKD1 antibody also can be derived from a “humanized” monoclonalantibody. Humanized monoclonal antibodies are produced by transferringmouse complementarity determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833, 1989,which is incorporated herein by reference. Techniques for producinghumanized monoclonal antibodies are described, for example, by Jones etal., Nature, 321:522, 1986; Riechmann et al., Nature 332:323, 1988;Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl.Acad. Sci. USA, 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992;and Singer et al., J. Immunol. 150:2844, 1993, which are incorporatedherein by reference.

Antibodies of the invention also can be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library (see, forexample, Barbas et al., Methods: A Companion to Methods in Enzymology,Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994,which are incorporated herein by reference). Cloning and expressionvectors that are useful for producing a human immunoglobulin phagelibrary can be obtained, for example, from Stratagene (La Jolla Calif.).

In addition, antibodies of the present invention can be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet., 7:13 (1994); Lonberg et al., Nature, 368:856(1994); Taylor et al., Int. Immunol., 6:579 (1994), each of which isincorporated herein by reference.

Antibody fragments of the invention can be prepared by proteolytichydrolysis of an antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, each of which inincorporated herein by reference (see, also, Nisonhoff et al., Arch.Biochem. Biophys, 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Meth. Enzymol. 1:422, 1967; and Coligan et al., atsections 2.8.1-2.8.10 and 2.10.1-2.10.4). Other methods of cleavingantibodies, such as separation of heavy chains to form monovalentlight-heavy chain fragments, further cleavage of fragments, or otherenzymatic, chemical, or genetic techniques can also be used, providedthe fragments bind to the antigen that is recognized by the intactantibody.

Fv fragments comprise an association of V_(H) and V_(L) chains, forexample, which can be noncovalent (see Inbar et al., Proc. Natl. Acad.Sci. USA 69:2659, 1972). The variable chains also can be linked by anintermolecular disulfide bond, can be crosslinked by a chemical such asglutaraldehyde (Sandhu, supra, 1992), or F_(v) fragments comprisingV_(H) and V_(L) chains can be connected by a peptide linker. Thesesingle chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., Methods: A Companion to Meth. Enzymol., 2:97, 1991; Bird et al.,Science 242:423, 1988; Ladner et al., U.S. Pat. No. 4,946,778; Pack etal., BioTechnology 11:1271, 1993; and Sandhu, supra, 1992).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Meth. Enzymol., 2:106, 1991).

A variety of methods can be employed utilizing reagents such as a mutantPKD1 polynucleotide, or oligonucleotide portion thereof and antibodiesdirected against a mutant PKD1 polypeptide or peptide. Specifically,such reagents can be used for the detection of the presence of PKD1mutations, e.g., molecules present in diseased tissue but absent from,or present in greatly reduced levels compared or relative to thecorresponding non-diseased tissue.

The methods described herein can be performed, for example, by utilizingpre-packaged kits, which can be diagnostic kits, comprising at least onespecific oligonucleotide portion of a PKD1 gene or mutant PKD1polynucleotide, a primer pair, or an anti-PKD1 antibody reagent asdisclosed herein, which can be conveniently used, for example, in aclinical setting to diagnose subjects exhibiting PKD1 abnormalities orto detect PKD1-associated disorders, including ADPKD. Any tissue inwhich a PKD1 polynucleotide is expressed can be utilized in a diagnosticmethod of the invention.

Nucleic acids from a tissue to be analyzed can be isolated usingprocedures that are well known in the art, or a diagnostic procedurescan be performed directly on a tissue section (fixed or frozen), whichcan be obtained from a subject by biopsy or resection, without furtherpurification. Oligonucleotide sequences of the invention can be used asprobes or primers for such in situ procedures (Nuovo, 1992, PCR in situhybridization: protocols and applications, Raven Press, N.Y.). Forexample, oligonucleotide probes useful in the diagnostic methods of theinvention include nucleotide sequences having at least 10 contiguousnucleotides and having a sequence substantially identical to a portionof SEQ ID NO:1, and including nucleotide 474, wherein nucleotide 474 isa T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110,wherein nucleotide 3110 is a C; nucleotide 8298, wherein nucleotide 8298is a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide9213, wherein nucleotide 9213 is an A; nucleotide 9326, whereinnucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is a T;nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide 10143,wherein nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide10234 is a C; nucleotide 10255, wherein nucleotide 10255 is a T, or acombination thereof. Primers useful in the present invention includethose set forth in SEQ ID NOS:3 to 18 and SEQ ID NOS: 19 to 51 and 61 to112. Such primers flank and can be used to amplify sequences containingone or more mutated nucleotides of a mutant PKD1 polynucleotide.

PKD1 polynucleotide sequences, either RNA or DNA, can be used inhybridization or amplification assays of biological samples to detectabnormalities of PKD1 expression; e.g., Southern or northern blotanalysis, single stranded conformational polymorphism (SSCP) analysisincluding in situ hybridization assays, or polymerase chain reactionanalyses, including detecting abnormalities by a methods such asdenaturing high performance liquid chromatography (DHPLC; also referredto as temperature-modulated heteroduplex chromatography) or conformationsensitive gel electrophoresis (CSGE), both of which are readilyadaptable to high throughput analysis (see, for example, Kristensen etal., BioTechniques 30:318-332, 2001; Leung et al., BioTechniques30:334-340, 2001, which are incorporated herein by reference). Suchanalyses can reveal quantitative abnormalities in the expression patternof the PKD1 polynucleotide, and, if the PKD1 mutation is, for example,an extensive deletion, or the result of a chromosomal rearrangement, canreveal more qualitative aspects of the PKD1 abnormality.

Diagnostic methods for detecting a mutant PKD1 polynucleotide caninvolve, for example, contacting and incubating nucleic acids derivedfrom a tissue sample being analyzed, with one or more labeledoligonucleotide probes of the invention or with a primer or primer pairof the invention, under conditions favorable for the specific annealingof these reagents to their complementary sequences within the targetmolecule. After incubation, non-annealed oligonucleotides are removed,and hybridization of the probe or primer, if any, to a nucleic acid fromthe target tissue is detected. Using such a detection scheme, the targettissue nucleic acid can be immobilized, for example, to a solid supportsuch as a membrane, or a plastic surface such as that on a microtiterplate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents are easily removed.Detection of the remaining, annealed, labeled nucleic acid reagents isaccomplished using standard techniques well known to those in the art.

Oligonucleotide probes or primers of the invention also can beassociated with a solid matrix such as a chip in an array, thusproviding a means for high throughput methods of analysis.Microfabricated arrays of large numbers of oligonucleotide probes (DNAchips) are useful for a wide variety of applications. Accordingly,methods of diagnosing or detecting a PKD1 variant or mutant can beimplemented using a DNA chip for analysis of a PKD1 polynucleotide anddetection of mutations therein. A methodology for large scale analysison DNA chips is described by Hacia et al. (Nature Genet. 14:441-447,1996; U.S. Pat. No. 6,027,880, which are incorporated herein byreference; see, also, Kristensen et al., supra, 2001). As described inHacia et al., high density arrays of over 96,000 oligonucleotides, eachabout 20 nucleotides in length, are immobilized to a single glass orsilicon chip using light directed chemical synthesis. Contingent on thenumber and design of the oligonucleotide probe, potentially every basein a sequence can be examined for alterations.

Polynucleotides or oligonucleotides applied to a chip can containsequence variations, which can be used to identify mutations that arenot yet known to occur in the population, or they can only thosemutations that are known to occur, including those disclosed herein (seeExample 2). Examples of oligonucleotides that can be applied to the chipinclude oligonucleotides containing at least 10 contiguous nucleotidesand having a sequence substantially identical to a portion of SEQ IDNO:1, and including nucleotide 474, wherein nucleotide 474 is a T;nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110, whereinnucleotide 3110 is a C; a position corresponding to nucleotide 3336,wherein nucleotide 3336 is deleted; nucleotide 3707, wherein nucleotide3707 is an A; nucleotide 4168, wherein nucleotide 4168 is a T;nucleotide 4885, wherein nucleotide 4885 is an A; nucleotide 5168,wherein nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058is a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide6089, wherein nucleotide 6089 is a T; nucleotide 6195, whereinnucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326 is aT; a position corresponding to nucleotides 7205 to 7211, whereinnucleotides 7205 to 7211 are deleted; nucleotide 7376, whereinnucleotide 7376 is a C; a nucleotide sequence corresponding tonucleotides 7535 to 7536, wherein a GCG nucleotide sequence is insertedbetween nucleotides 7535 and 7536; nucleotide 7415, wherein nucleotide7415 is a T; nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide7696, wherein nucleotide 7696 is a T; nucleotide 7883, whereinnucleotide 7883 is a T; nucleotide 8021, wherein nucleotide 8021 is anA; a nucleotide sequence corresponding to nucleotide 8159 to 8160,wherein nucleotides 8159 to 8160 are deleted; nucleotide 8298, whereinnucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G;nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326,wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367is a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide10143, wherein nucleotide 10143 is a G; nucleotide 10234, whereinnucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255 is aT; or a combination thereof.

Prior to hybridization with oligonucleotide probes on the chip, the testsample is isolated, amplified and labeled (e.g. fluorescent markers).The test polynucleotide sample is then hybridized to the immobilizedoligonucleotides. The intensity of sequence-based techniques of thetarget polynucleotide to the immobilized probe is quantitated andcompared to a reference sequence. The resulting genetic information canbe used in molecular diagnosis. A common utility of the DNA chip inmolecular diagnosis is screening for known mutations.

In addition to DNA chip methodology, methods using machinery adapted toDNA analysis can allow for commercialization of the disclosed methods ofdetection of PKD1 mutations and diagnosis of ADPKD. For example,genotyping by mass spectrometry can be used, or matrix-assisted laserdesorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry canbe used for mass genotyping of single-base pair and short tandem repeatmutant and variant sequences. For example, PCR amplification of theregion of the mutation with biotin attached to one of the primers can beconducted, followed by immobilization of the amplified DNA tostreptavidin beads. Hybridization of a primer adjacent to the variant ormutant site is performed, then extension with DNA polymerase past thevariant or mutant site in the presence of dNTPs and ddNTPs is performed.When suitably designed according to the sequence, this results in theaddition of only a few additional bases (Braun, Little, Koster, 1997).The DNA is then processed to remove unused nucleotides and salts, andthe short primer plus mutant site is removed by denaturation andtransferred to silicon wafers using a piezoelectric pipette. The mass ofthe primer+variant or mutant site is then determined by delayedextraction MALDI-TOF mass spectrometry. Single base pair and tandemrepeat variations in sequence are easily determined by their mass. Thisfinal step is very rapid, requiring only 5 sec per assay, and all ofthese steps can be automated, providing the potential of performing upto 20,000 genotypings per day. This technology is rapid, extremelyaccurate, and adaptable to any variant or mutation, can identify bothsingle base pair and short tandem repeat variants, and adding orremoving variant or mutant sequences to be tested can be done in a fewseconds at trivial cost.

Another diagnostic methods for the detection of mutant PKD1polynucleotides involves amplification, for example, by PCR (see U.S.Pat. No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad.Sci. USA 88:189-193, 1991a), self sustained sequence replication(Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990),transcriptional amplification system (Kwoh et al., Proc. Natl. Acad.Sci. USA 86:1173-1177, 1989), Q-Beta Replicase (Lizardi et al.,Bio/Technology 6:1197, 1988), or any other RNA amplification method,followed by the detection of the amplification products. The presentinvention provides reagents, methods and compositions that can be usedto overcome prior difficulties with diagnosing ADPKD.

Using the primer pairs and methods described herein, the entirereplicated segment of the PKD1 gene, including exons 1 and 22, can beamplified from genomic DNA to generate a set of eight long rangeamplification products, which range in size from about 0.3 kb to 5.8 kb(Table 1; see, also, FIG. 1). The availability of widely scatteredPKD1-specific primers provides a means to anchor PKD1-specificamplification, and the ability to use various primer combinationsprovides a means to produce longer or shorter amplification products asdesired. For example, the largest PKD1 fragment, which is amplified byprimers BPF13 and KG8R25 (see Table 1; SEQ ID NOS: 17 and 18,respectively), can be divided into two shorter segments by using thePKD1-specific primer, KG85R25 (SEQ ID NO:18), with forward nested primerF32 (5′-GCCTTGCGCAGCTTGGACT-3′; SEQ ID NO:53), and using BPF13 (SEQ IDNO:17) and a second specific primer, 31R (5′-ACAGTGTCTTGAGTCCAAGC-3′;SEQ ID NO:54).

It should be recognized that, while many of the primers disclosed hereinare positioned with intronic sequences of the PKD1 gene, others such asSEQ ID NO:16 are positioned in coding sequences. As such, a cDNAmolecule can obtained from a target RNA molecule, for example, byreverse transcription of the RNA molecule using a primer such as SEQ IDNO:16 and an appropriate second primer positioned 5′ or 3′ to SEQ IDNO:16. In this embodiment, a PKD1 RNA can be isolated from any tissue inwhich wild type PKD1 is known to be expressed, including, for example,kidney tissue, nucleated peripheral blood cells, and fibroblasts. Atarget sequence within the cDNA is then used as the template for anucleic acid amplification reaction, such as a PCR amplificationreaction, or the like. An amplification product can be detected, forexample, using radioactively or fluorescently labeled nucleotides or thelike and an appropriate detection system, or by generating a sufficientamount of the amplification product such that it can be visualized byethidium bromide staining and gel electrophoresis.

Genomic DNA from a subject, including from a cell or tissue sample, canbe used as the template for generating a long range PKD1-specificamplification product. Methods of isolating genomic DNA are well knownand routine (see Sambrook et al., supra, 1989). Amplification of thegenomic PKD1 DNA has advantages over the cDNA amplification process,including, for example, allowing for analysis of exons and introns ofthe PKD1 gene. As such, a target sequence of interest associated witheither an intron or exon sequence of a PKD1 gene can be amplified andcharacterized. A target sequence of interest is any sequence or locus ofa PKD1 gene that contains or is thought to contain a mutation, includingthose mutations that correlate to a PKD1-associated disorder or disease.

Using primers flanking the target sequence, a sufficient number of PCRcycles is performed to provide a PKD1-specific amplification productcorresponding to the target sequence. If desired, additionalamplification can be performed, for example, by performing a nested PCRreaction. Examples of primers useful for generating a PKD1-specificfirst amplification product from genomic DNA include the primer pairshaving sequences as exemplified in SEQ ID NO:3 and 4; SEQ ID NOS:5 and6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ IDNOS:13 and 14; SEQ ID NOS:15 and 16; and SEQ ID NOS:17 and 18. ThePKD1-specific first amplification product can be further amplified usingnested primers specific for a target sequence, including the primerpairs exemplified as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ IDNOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42;SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ IDNOS:49 and 50; SEQ ID NOS:51 and 61; and the primer pairs formed usingconsecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96, 113,and 97 to 112.

The amplified target sequences can be examined for changes (i.e.,mutations) with respect to SEQ ID NO:1 using any of various well knownmethods as disclosed herein or otherwise known in the art. For example,the amplification products can simply be sequenced using routine DNAsequencing methods, particularly where only one or few amplificationproducts are to be examined. However, DNA sequencing will be morevaluable as a method of detecting mutations according to a method of theinvention as sequencing technology improves and becomes more adaptableto high throughput screening assays. In addition, methods that areuseful for detecting the presence of a mutation in a DNA sequenceinclude, for example, DHPLC (Huber et al., Nucl. Acids Res.21:1061-10666, 1993; Liu et al., Nucl. Acids Res. 26:1396-1400, 1998;Choy et al., Ann. Hum. Genet. 63:383-391, 1999; Ellis et al., Hum.Mutat. 15:556-564, 2000; which are incorporated herein by reference;see, also, Kristensen et al., supra, 2001); CSGE (Leung et al., supra,2001); single-stranded conformation analysis (SSCA; Orita et al., Proc.Natl. Acad. Sci., USA 86:2766-2770, 1989); denaturing gradient gelelectrophoresis (DGGE; Sheffield et al., Proc. Natl. Acad. Sci., USA86:232-236, 1989); RNase protection assays; allele-specificoligonucleotides (ASOs; Handelin and Shuber, Current Protocols in HumanGenetics, Suppl. 16 (John Wiley & Sons, Inc. 1998), 9:9.4.1-9.4.8); theuse of proteins that recognize nucleotide mismatches, such as the E.coli mutS protein; and allele-specific PCR.

For allele-specific PCR, primers are used that hybridize at their 3′ends to a particular mutations. Examples of primers that can be used forallele specific PCR include an oligonucleotide of at least 10 nucleotideof SEQ ID NO:1 and that has at its 3′ end nucleotide 474, whereinnucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an A;nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide 8298,wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164is a G; nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide9326, wherein nucleotide 9326 is a T; nucleotide 9367, whereinnucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is anA; nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234,wherein nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide10255 is a T. If the particular mutation is not present, anamplification product is not observed. Amplification Refractory MutationSystem (ARMS) can also be used (see European Patent Application Publ.No. 0332435; Newton et al., Nucl. Acids. Res. 17:2503-2516, 1989).

In the SSCA, DGGE and RNase protection methods, a distinctiveelectrophoretic band appears. SSCA detects a band that migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned that detects a specific sequence, and the assay is performed bydetecting the presence or absence of a hybridization signal. In the mutSassay, the protein binds only to sequences that contain a nucleotidemismatch in a heteroduplex between mutant and wild-type sequences.

Denaturing gradient gel electrophoresis is based on the melting behaviorof the DNA fragments and the use of denaturing gradient gelelectrophoresis as shown by Fischer and Lerman, Proc. Natl. Acad. Sci.USA 80:1579-83, 1983; Myers et al.; Nucl. Acids Res. 13:3111-3129, 1985;Lerman et al., in Molecular Biol. of Homo Sapiens, Cold Spring HarborLab. (1986) pp. 285-297. DNA fragments differing by single basesubstitutions can be separated from each other by electrophoresis inpolyacrylamide gels containing an ascending gradient of the DNAdenaturants urea and formamide. Two identical DNA fragments differing byonly one single base pair, will initially move through thepolyacrylamide gel at a constant rate. As they migrate into a criticalconcentration of denaturant, specific domains within the fragments meltto produce partially denatured DNA. Melting of a domain is accompaniedby an abrupt decrease in mobility. The position in the denaturantgradient gel at which the decrease in mobility is observed correspondsto the melting temperature of that domain. Since a single basesubstitution within the melting domain results in a melting temperaturedifference, partial denaturation of the two DNA fragments will occur atdifferent positions in the gel. DNA molecules can therefore be separatedon the basis of very small differences in the melting temperature.Additional improvements to this DGGE have been made as disclosed byBorresen in U.S. Pat. No. 5,190,856. In addition, after a first DGGEanalysis, an identified product can be cloned, purified and analyzed asecond time by DGGE.

Denaturing high performance liquid chromatography (DHPLC; Kristensen etal., supra, 2001) and high throughput conformation sensitive gelelectrophoresis (HTCSGD; Leung et al., supra, 2001) are particularlyuseful methods for detecting a mutant PKD1 polynucleotide sequencebecause the methods are readily adaptable to high throughput analysis.In addition, these methods are suitable for detecting known mutations aswell as identifying previously unknown mutations. As such, these methodsof detection can be adopted for use in clinical diagnostic settings.DHPLC, for example, can be used to rapidly screen a large number ofsamples, for example, 96 samples prepared using a 96 well microtiterplate format, to identify those showing a change in the denaturationproperties. Where such a change is identified, confirmation that thePKD1 polynucleotide in the sample showing the altered denaturationproperty is a mutant PKD1 polynucleotide can be confirmed by DNAsequence analysis, if desired.

An oligonucleotide probe specific for a mutant PKD1 polynucleotide alsocan be used to detect a mutant PKD1 polynucleotide in a biologicalsample, including in a biological fluid, in cells or tissues obtainedfrom a subject, or in a cellular fraction such as an organellepreparation. Cellular sources useful as samples for identifying a mutantPKD1 polynucleotide include, for example, renal cells including renaltubular epithelial cells, bile duct cells, skeletal muscle cells, lungalveolar epithelial cells, placental cells, fibroblasts and lymphocytes.Biological fluids useful as samples for identifying a mutant PKD1polynucleotide include, for example, whole blood or serum or plasmafractions, urine, mucous, and saliva. A biological sample such as atissue or cell sample can be obtained by any method routinely used in aclinical setting, including, for example, by cellular aspiration, biopsyor other surgical procedure.

The oligonucleotide probe can be labeled with a compound that allowsdetection of binding to a mutant PKD1 polynucleotide in the sample. Adetectable compound can be, for example, a radioactive label, whichprovides a highly sensitive means for detection, or a non-radioactivelabel such as a fluorescent, luminescent, chemiluminescent, orenzymatically detectable label or the like (see, for example, Matthewset al., Anal. Biochem. 169:1-25, 1988).

The method of detection can be a direct or indirect method. An indirectdetection process can involve, for example, the use of anoligonucleotide probe that is labeled with a hapten or ligand such asdigoxigenin or biotin. Following hybridization, the target-probe duplexis detected by the formation of an antibody or streptavidin complex,which can further include an enzyme such as horseradish peroxidase,alkaline phosphatase, or the like. Such detection systems can beprepared using routine methods, or can be obtained from a commercialsource. For example, the GENIUS detection system (Boehringer Mannheim)is useful for mutational analysis of DNA, and provides an indirectmethod using digoxigenin as a tag for the oligonucleotide probe and ananti-digoxigenin-antibody-alkaline phosphatase conjugate as the reagentfor identifying the presence of tagged probe.

Direct detection methods can utilize, for example, fluorescent labeledoligonucleotides, lanthanide chelate labeled oligonucleotides oroligonucleotide-enzyme conjugates. Examples of fluorescent labelsinclude fluorescein, rhodamine and phthalocyanine dyes. Examples oflanthanide chelates include complexes of europium (Eu³⁺) or terbium(Tb³⁺). Oligonucleotide-enzyme conjugates are particularly useful fordetecting point mutations when using target-specific oligonucleotides,as they provide very high sensitivities of detection.Oligonucleotide-enzyme conjugates can be prepared by a number of methods(Jablonski et al., Nucl. Acids Res. 14:6115-6128, 1986; Li et al., Nucl.Acids. Res. 15:5275-5287, 1987; Ghosh et al., Bioconjugate Chem.1:71-76, 1990). The detection of target nucleic acids using theseconjugates can be carried out by filter hybridization methods or bybead-based sandwich hybridization (Ishii et al., Bioconjugate Chem.4:34-41, 1993).

Methods for detecting a labeled oligonucleotide probe are well known inthe art and will depend on the particular label. For radioisotopes,detection is by autoradiography, scintillation counting or phosphorimaging. For hapten or biotin labels, detection is with antibody orstreptavidin bound to a reporter enzyme such as horseradish peroxidaseor alkaline phosphatase, which is then detected by enzymatic means. Forfluorophor or lanthanide chelate labels, fluorescent signals can bemeasured with spectrofluorimeters, with or without time-resolved mode orusing automated microtiter plate readers. For enzyme labels, detectionis by color or dye deposition, for example, p-nitrophenyl phosphate or5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for alkalinephosphatase, and 3,3′-diaminobenzidine-NiCl₂ for horseradish peroxidase,fluorescence by 4-methyl umbelliferyl phosphate for alkalinephosphatase, or chemiluminescence by the alkaline phosphatase dioxetanesubstrates LumiPhos 530 (Lumigen Inc., Detroit Mich.) or AMPPD and CSPD(Tropix, Inc.). Chemiluminescent detection can be carried out with X-rayor Polaroid film, or by using single photon counting luminometers, whichalso is a useful detection format for alkaline phosphatase labeledprobes.

Mutational analysis can also be carried out by methods based on ligationof oligonucleotide sequences that anneal immediately adjacent to eachother on a target DNA or RNA molecule (Wu and Wallace, Genomics4:560-569, 1989; Landren et al., Science 241:1077-1080, 1988; Nickersonet al., Proc. Natl. Acad. Sci. USA 87:8923-8927, 1990; Barany, supra,1991a). Ligase-mediated covalent attachment occurs only when theoligonucleotides are correctly base-paired. The ligase chain reaction(LCR) and the oligonucleotide ligation assay (OLA), which utilize thethermostable Taq ligase for target amplification, are particularlyuseful for interrogating mutation loci. The elevated reactiontemperatures permit the ligation reaction to be conducted with highstringency (Barany, PCR Methods and Applications 1:5-16, 1991b; Grossmanet al., Nucl. Acids. Res. 22:4527-4534, 1994, which are incorporatedherein by reference).

Analysis of point mutations in DNA can also be carried out by using PCRand variations thereof. Mismatches can be detected by competitiveoligonucleotide priming under hybridization conditions where binding ofthe perfectly matched primer is favored (Gibbs et al., Nucl. Acids. Res.17:2437-2448, 1989). In the amplification refractory mutation systemtechnique (ARMS), primers can be designed to have perfect matches ormismatches with target sequences either internal or at the 3′ residue(Newton et al., supra, 1989). Under appropriate conditions, only theperfectly annealed oligonucleotide can function as a primer for the PCRreaction, thus providing a method of discrimination between normal andmutant sequences.

Detection of single base mutations in target nucleic acids can beconveniently accomplished by differential hybridization techniques usingsequence-specific oligonucleotides (Suggs et al., Proc. Natl. Acad. Sci.USA 78:6613-6617, 1981; Conner et al., Proc. Natl. Acad. Sci. USA80:278-282, 1983; Saiki et al., Proc. Natl. Acad. Sci. USA 86:6230-6234,1989). Mutations can be diagnosed on the basis of the higher thermalstability of the perfectly matched probes as compared to the mismatchedprobes. The hybridization reactions can be carried out in a filter-basedformat, in which the target nucleic acids are immobilized onnitrocellulose or nylon membranes and probed with oligonucleotideprobes. Any of the known hybridization formats can be used, includingSouthern blots, slot blots, reverse dot blots, solution hybridization,solid support based sandwich hybridization, bead-based, siliconchip-based and microtiter well-based hybridization formats.

An alternative strategy involves detection of the mutant sequences bysandwich hybridization methods. In this strategy, the mutant and wildtype target nucleic acids are separated from non-homologous DNA/RNAusing a common capture oligonucleotide immobilized on a solid supportand detected by specific oligonucleotide probes tagged with reporterlabels. The capture oligonucleotides can be immobilized on microtiterplate wells or on beads (Gingeras et al., J. Infect. Dis. 164:1066-1074,1991; Richen et al., Proc. Natl. Acad. Sci. USA 88:11241-11245, 1991).

Another method for analysis of a biological sample for specificmutations in a PKD1 polynucleotide sequence (e.g., mutant PKD1polynucleotides, or oligonucleotide portions thereof) is a multiplexedprimer extension method. In this method primer is hybridized to anucleic acid suspected of containing a mutation such that the primer ishybridized 3′ to the suspected mutation. The primer is extended in thepresence of a mixture of one to three deoxynucleoside triphosphates andone of three chain terminating deoxynucleoside triphosphates selectedsuch that the wild-type extension product, the mutant DNA-derivedextension product and the primer each are of different lengths. Thesesteps can be repeated, such as by PCR or RT-PCR, and the resultingprimer extended products and primer are then separated on the basis ofmolecular weight to thereby enable identification of mutant DNA-derivedextension product.

In one aspect of the invention, the OLA is applied for quantitativemutational analysis of PKD1 polynucleotide sequences (Grossman, et al.,supra, 1994). In this embodiment of the invention, a thermostableligase-catalyzed reaction is used to link a fluorescently labeled commonprobe with allele-specific probes. The latter probes are sequence-codedwith non-nucleotide mobility modifiers that confer uniqueelectrophoretic mobilities to the ligation products.

Oligonucleotides specific for wild type or mutant PKD1 sequences can besynthesized with different oligomeric nucleotide or non-nucleotidemodifier tails at their 5′ termini. Examples of nucleotide modifiers areinosine or thymidine residues, whereas examples of non-nucleotidemodifiers include pentaethyleneoxide (PEO) and hexaethyleneoxide (HEO)monomeric units. The non-nucleotide modifiers are preferred and mostpreferably PEO is used to label the probes. When a DNA template ispresent, a thermostable DNA ligase catalyzes the ligation of normal andmutant probes to a common probe bearing a fluorescent label. The PEOtails modify the mobilities of the ligation products in electrophoreticgels. The combination of PEO tails and fluorophor labels (TET and FAM(5-carboxy-fluorescein derivatives)), HEX and JOE (6-carboxy-fluoresceinderivatives), ROX (6-carboxy-x-rhodamine), or TAMRA(N,N,N′,N′-tetramethyl-6-carboxy-rhodamine; Perkin-Elmer, ABI Division,Foster City Calif.) allow multiplex analysis based on size and color byproviding unique electrophoretic signatures to the ligation products.The products are separated by electrophoresis, and fluorescenceintensities associated with wild type and mutant products are used toquantitate heteroplasmy. Thus, wild type and mutant, including variant,sequences are detected and quantitated on the basis of size andfluorescence intensities of the ligation products. This method furthercan be configured for quantitative detection of multiple PKD1polynucleotide mutations in a single ligation reaction.

Mismatch detection or mutation analysis can also be performed usingmismatch specific DNA intercalating agents. Such agents intercalate at asite having a mismatch followed by visualization on a polyacrylamide oragarose gel or by electrocatalysis. Accordingly, PKD1 polynucleotidesequences can be contacted with probes specific for a PKD1 mutation orprobes that are wild type for an area having a specific mutation underconditions such that the PKD1 polynucleotide and probe hybridize. Thehybridized sequences are then contacted with a mismatch intercalatingagent and, for example, separated on a gel. Visualized bands on the gelcorrespond to a sequence having a mismatch. If the probes are wild-typeprobes mismatches will occur if the target PKD1 sequence contains amismatch. If the probes are specific for a mutated sequence mismatcheswill be present where the target PKD1 sequence is wild type, but thehybridized or duplex sequences will not contain mismatches where theprobe sequence hybridizes to a PKD1 sequence containing the samemutation.

For quantitative analysis of PKD1 mutations using OLA, oligonucleotideprobes are preferably labeled with fluorophor labels that providespectrally distinguishable characteristics. In one embodiment,oligonucleotides are labeled with 5′ oligomeric PEO tails. Synthesis ofsuch 5′ labeled oligonucleotides can be carried out, for example, usingan automated synthesizer using standard phosphoramidite chemistry.Following cleavage from resin and deprotection with ammonium hydroxide,the (PEO)_(n)-oligonucleotides can be purified by reverse phase HPLC.Oligonucleotides with 3′-FAM or TET dyes (Perkin Elmer) and5′-phosphates can be synthesized and purified by the procedure ofGrossman et al., supra, 1994. The 5′-PEO-labeled probes can besynthesized to have 5′-PEO-tails of differing lengths to facilitatedistinguishing the ligated probe products both electrophoretically bysize and by spectral characteristics of the fluorophor labels.

The oligonucleotide probes are used for identifying mutant PKD1polynucleotides, which can be indicative of a PKD1-associated disordersuch as ADPKD. Preferably, the probes are specific for one or more PKD1nucleotide positions of SEQ ID NO:1 selected from nucleotide 474,wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 isan A; nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide 8298,wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164is a G; nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide9326, wherein nucleotide 9326 is a T; nucleotide 9367, whereinnucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064 is anA; nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide 10234,wherein nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide10255 is a T. The oligonucleotide probes for the OLA assay are typicallydesigned to have calculated melting temperatures of about 40° C. to 50°C., generally about 48° C., by the nearest neighbor method (Breslaur etal., Proc. Natl. Acad. Sci. USA 83:9373-9377, 1986) so that the ligationreaction can be performed at a temperature range of about 40° C. to 60°C., typically from about 45° C. to about 55° C. The wild type andmutant, including variant, oligonucleotide probes can be synthesizedwith various combinations of PEO oligomeric tails and fluorescein dyessuch as TET and FAM. These combinations of mobility modifiers andfluorophor labels furnish electrophoretically unique ligation productsthat can enable the monitoring of two or more PKD1 nucleotide sites in asingle ligation reaction.

In one embodiment, a method of diagnosing a PKD1-associated disorder ina subject is performed by amplifying a portion of a PKD1 polynucleotidein a nucleic acid sample from a subject suspected of having aPKD1-associated disorder with at least a first primer pair to obtain afirst amplification product, wherein said first primer pair is a primerpair of claim 3; amplifying the first amplification product with atleast a second primer pair to obtain a nested amplification product,wherein the second primer pair is suitable for performing nestedamplification of the first amplification product; and determiningwhether the nested amplification product has a mutation associated witha PKD1-associated disorder, wherein the presence of a mutationassociated with a PKD1-associated disorder is indicative of aPKD1-associated disorder, thereby diagnosing a PKD1-associated disorderin the subject. The method can be performed using a first primer pairselected from SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQID NOS:15 and 16; SEQ ID NOS:17 and 18; and a combination thereof, and asecond primer pair selected from SEQ ID NOS:19 and 20; SEQ ID NOS:21 and22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28;SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ IDNOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; SEQ ID NOS:62 and 63;SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ IDNOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID NOS:76and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81; SEQ ID NOS:82 and83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID NOS:88 and 89;SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ IDNOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ IDNOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and 106; SEQ IDNOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111 and 112; anda combination thereof.

In another embodiment, a method of diagnosing a PKD1-associated disorderin a subject is performed by amplifying a portion of PKD1 polynucleotidein a nucleic acid sample from a subject suspected of having aPKD1-associated disorder with a first primer pair to obtain a firstamplification product; amplifying the first amplification product usinga second primer pair to obtain a second amplification product; anddetecting a mutation in the second amplification product, wherein themutation comprises SEQ ID NO:1 wherein nucleotide 3110 is a C;nucleotide 3336 is deleted; nucleotide 3707 is an A; nucleotide 5168 isa T; nucleotide 6078 is an A; nucleotide 6089 is a T; nucleotide 6326 isa T; nucleotides 7205 to 7211 are deleted; nucleotide 7415 is a T;nucleotide 7433 is a T; nucleotide 7883 is a T; or nucleotides 8159 to8160 are deleted; nucleotide 8298 is a G; nucleotide 9164 is a G;nucleotide 9213 is an A; or nucleotide 9326 is a T; nucleotide 10064 isan A; or wherein a GCG nucleotide sequence is inserted betweennucleotides 7535 and 7536; or a combination thereof, thereby diagnosinga PKD1-associated disorder in the subject.

The present invention also provides a method of identifying a subjecthaving or at risk of having a PKD1-associated disorder. Such a methodcan be performed, for example, by comprising contacting nucleic acidmolecules in a sample from a subject with at least one primer pair ofthe invention under conditions suitable for amplification of a PKD1polynucleotide by the primer pair, thereby generating a PKD1-specificamplification product; and testing an amplification product for thepresence or absence of a mutation indicative of a PKD1-associateddisorder, wherein the absence of the mutation identifies the subject anot having or at risk of the having a PKD1-associated disorder, andwherein the presence of the mutation identifies the subject as having oris at risk of having a PKD1-associated disorder. The primer pair can be,for example, selected from SEQ ID NO:3 and 4; SEQ ID NO:5 and 6; SEQ IDNOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18. ThePKD1-associated disorder can be autosomal dominant polycystic kidneydisease, acquired cystic disease, or any other PKD-1 associateddisorder, and the subject can be, for example, a vertebrate,particularly a human subject.

Such a method is particularly adaptable to a high throughput format,and, if desired, can include a step of contacting the PKD1-specificamplification product with at least a second primer pair, underconditions suitable for nested amplification of the PKD1-specificamplification product by a second primer pair, thereby generating anested amplification product, then testing the nested amplificationproduct for the presence or absence of a mutation indicative of aPKD1-associated disorder. The second primer pair can be any primer pairsuitable for nested amplification of the PKD1-specific amplificationproduct, for example, a primer pair selected from SEQ ID NOS:19 and 20;SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ IDNOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46;SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; SEQ IDNOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81;SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ IDNOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94and 95; SEQ ID NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and100; SEQ ID NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and106; SEQ ID NOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111and 112; and a combination thereof.

Testing an amplification product for the presence or absence of themutation can be performed using any of various well known methods forexamining a nucleic acid molecule. For example, nucleotide sequence ofthe amplification product can be determined, and compared with thenucleotide sequence of a corresponding nucleotide sequence of SEQ IDNO:1. The amplification product also can be tested by determining themelting temperature of the amplification product, and comparing themelting temperature to the melting temperature of a correspondingnucleotide sequence of SEQ ID NO:1. The melting temperature can bedetermined, for example, using denaturing high performance liquidchromatography.

Where a nested amplification is to be performed, the method can includea step directed to reducing contamination of the PKD1-specificamplification product by genomic DNA prior to contacting thePKD1-specific amplification product with the at least second set ofprimer pairs. For example, contamination of the PKD1-specificamplification product can be reduced by diluting the PKD1-specificamplification product.

The mutation indicative of a of PKD1 associated disorder can be, forexample, a nucleotide sequence substantially identical to SEQ ID NO:1,wherein nucleotide 3110 is a C; nucleotide 8298 is a G; nucleotide 9164is a G; nucleotide 9213 is an A; nucleotide 9326 is a T; or nucleotide10064 is an A; or can be a nucleotide sequence substantially identicalto SEQ ID NO:1, wherein nucleotide 3336 is deleted; nucleotide 3707 isan A; nucleotide 5168 is a T; nucleotide 6078 is an A; nucleotide 6089is a T; nucleotide 6326 is a T; nucleotides 7205 to 7211 are deleted;nucleotide 7415 is a T; nucleotide 7433 is a T; nucleotide 7883 is a T;or nucleotides 8159 to 8160 are deleted; or wherein a GCG nucleotidesequence is inserted between nucleotides 7535 and 7536.

Data that is collected pursuant to a step of detecting the presence orabsence of a mutation indicative of a PKD1-associated disorder in anamplification product, which can be an amplification product generatedaccording to a method of the invention, including, for example, aPKD1-specific amplification product or a nested amplification product,can be accumulated, and can be formatted into a form that facilitatesdetermining, for example, whether a subject is at risk of aPKD1-associated disorder. As such, the data can be formatted into areport that indicates whether a subject is at risk of a PKD1-associatedisorder. The report can be in any of various forms, including, forexample, contained in a computer random access or read-only memory, orstored on a diskette, CD, DVD, magnetic tape; presented on a visualdisplay such as a computer monitor or other cathode ray tube or liquidcrystal display; or printed on paper. Furthermore, the data, which canbe formatted into a report, can be transmitted to a user interested inor privy to the information. The data or report can be transmitted usingany convenient medium, for example, via the internet, by facsimile or bymail, depending on the form of the data or report.

Also provided is a method of detecting the presence of a mutant PKD1polynucleotide in a sample by contacting a sample suspected ofcontaining a mutant PKD1 polynucleotide with an oligonucleotide of theinvention under conditions that allow the oligonucleotide to selectivelyhybridize with a mutant PKD1 polynucleotide; and detecting selectivehybridization of the oligonucleotide and a mutant PKD1 polynucleotide,thereby detecting the presence of a mutant PKD1 polynucleotide sequencein the sample. In another embodiment, a method of detecting the presenceof a mutant PKD1 polypeptide in a sample is provided, for example, bycontacting a sample suspected of containing a mutant PKD1 polypeptidewith an antibody of the invention under conditions that allow theantibody to specifically bind a mutant PKD1 polypeptide; and detectingspecific binding of the antibody and the mutant PKD1 polypeptide in thesample, thereby detecting the presence of a mutant PKD1 polypeptide in asample. The mutant PKD1 polypeptide can have a sequence, for example,substantially as set forth in SEQ ID NO:2, and having a mutation ofA88V, W967R, L2696R, R2985G, W3001X, R3039C, V3285I, H3311R, or acombination thereof (see, also, Table 4).

Antibodies that can specifically bind wild type or mutant PKD1polypeptides, or peptide portions thereof, can also be used as ADPKDdiagnostic reagents. Such reagents provide a diagnostic method that candetect the expression of abnormal PKD1 proteins or of abnormal levels ofPKD1 protein expression, including the detection of mutant PKD1polypeptides or aberrant cellular localization of a PKD1 protein. Forexample, differences in the size, electronegativity, or antigenicity ofthe mutant PKD1 protein relative to a wild type PKD1 protein can bedetected.

Diagnostic methods for the detection of mutant PKD1 polypeptides orpeptide portions thereof can involve, for example, immunoassays whereinepitopes of a mutant PKD1 polypeptide are detected by their interactionwith an anti-PKD1 specific antibody (e.g., an anti-mutant PKD1 specificantibody). For example, an antibody that specifically binds to a mutantPKD1 polypeptide does not bind to a wild-type PKD1 polypeptide orpeptide thereof. Particular epitopes of PKD1 to which antibodies can bedeveloped include peptides that are substantially identical to SEQ IDNO:2, and having at least five amino acids, including amino acid residue88, wherein residue 88 is a V; residue 967, wherein residue 967 is an R;residue 2696, wherein residue 2696 is an R; residue 2985, whereinresidue 2985 is a G; residue 3039, wherein residue 3039 is a C; residue3285, wherein residue 3285 is an I; or residue 3311, wherein residue3311 is an R; or a C-terminal peptide including amino acid residue 3000,where residue 3001 is absent and the mutant PKD1 polypeptide istruncated due to the presence of a STOP codon in the encoding mutantPKD1 polynucleotide.

Antibodies, or fragments of antibodies, such as those described, above,are useful in the present invention and can be used to quantitatively orqualitatively detect the presence of wild type or mutant PKD1polypeptides or peptide portions thereof, for example. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection.

The antibodies (or fragments thereof) useful in the present inventioncan, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of PKD1 polypeptide,peptides, variants or mutants thereof. Detection can be accomplished byremoving a histological specimen from a subject, and applying thereto alabeled antibody of the present invention. The histological sample canbe taken from a tissue suspected of exhibiting ADPKD. The antibody (orfragment) is preferably applied by overlaying the labeled antibody (orfragment) onto a biological sample. Through the use of such a procedure,it is possible to determine not only the presence of PKD1 polypeptides,but also their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild type or mutant PKD1 polypeptide or peptideportions thereof typically comprise incubating a biological sample, suchas a biological fluid, a tissue extract, freshly harvested cells, orcells that have been incubated in tissue culture, in the presence of adetectably labeled antibody capable of identifying a PKD1 polypeptide,mutant PKD1 polypeptide and peptide portions thereof, and detecting thebound antibody by any of a number of techniques well-known in the art.The biological sample can be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled mutant PKD1 specificantibody, preferably an antibody that recognizes a developed includepeptides that are substantially identical to SEQ ID NO:2, and having atleast five amino acids, including amino acid residue 88, wherein residue88 is a V; residue 967, wherein residue 967 is an R; residue 2696,wherein residue 2696 is an R; residue 2985, wherein residue 2985 is a G;residue 3039, wherein residue 3039 is a C; residue 3285, wherein residue3285 is an I; or residue 3311, wherein residue 3311 is an R; or aC-terminal peptide including amino acid residue 3000, where residue 3001is absent and the mutant PKD1 polypeptide is truncated due to thepresence of a STOP codon in the encoding mutant PKD1 polynucleotide(see, also, Table 4). The solid phase support can then be washed withthe buffer a second time to remove unbound antibody, and the amount ofbound label on solid support can be detected by conventional meansspecific for the label.

A “solid phase support” or “carrier” can be any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, or the like. Those skilled inthe art will know many other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of an anti-mutant PKD1 antibody canbe determined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation. One of the waysin which the mutant PKD1-specific antibody can be detectably labeled isby linking the antibody to an enzyme and use the enzyme labeled antibodyin an enzyme immunoassay (EIA; Voller, “The Enzyme Linked ImmunosorbentAssay (ELISA): Diagnostic Horizons 2:1-7, 1978; MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.); Voller et al., J.Clin. Pathol. 31:507-520, 1978; Butler, Meth. Enzymol. 73:482-523, 1981;Maggio (ed.), “Enzyme Immunoassay”, CRC Press, Boca Raton Fla., 1980;Ishikawa et al., (eds.), “Enzyme Immunoassay”, Kgaku Shoin, Tokyo,1981). The enzyme that is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorimetric or by visual means.

Enzymes that can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,1-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.In addition, detection can be accomplished using any of a variety ofother immunoassays, including, for example, by radioactively labelingthe antibodies or antibody fragments and detecting PKD1 wild type ormutant peptides using a radioimmunoassay (RIA; see, for example,Weintraub, Principles of Radioimmunoassays, Seventh Training Course onRadioligand Assay Techniques, The Endocrine Society, March, 1986, whichis incorporated herein by reference). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

The antibody also can be labeled with a fluorescent compound. When thefluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among themost commonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine. The antibody can also be detectablylabeled using fluorescence emitting metals such as ¹⁵²Eu, or others ofthe lanthanide series. These metals can be attached to the antibodyusing such metal chelating groups as diethylenetriaminepentacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound can be used to labelthe antibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

In vitro systems can be designed to identify compounds capable ofbinding a mutant PKD1 polynucleotide of the invention (e.g., apolynucleotide having a sequence substantially identical to SEQ ID NO:1and having a mutation such as C474T; G487A; T3110C; T8298G; A9164G;G9213A; C9326T; C9367T; G10064A; A10143G; T10234C; or G10255T). Suchcompounds can include, but are not limited to, peptides made of D-and/or L-configuration amino acids in, for example, the form of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84, 1981),phosphopeptides in, for example, the form of random or partiallydegenerate, directed phosphopeptide libraries (see, e.g., Songyang etal., Cell 72:767-778, 1993), antibodies, and small or large organic orinorganic molecules. Compounds identified can be useful, for example, inmodulating the activity of PKD1 proteins, variants or mutants. Forexample, mutant PKD1 polypeptides of the invention can be useful inelaborating the biological function of the PKD1 protein. Such mutantscan be utilized in screens for identifying compounds that disrupt normalPKD1 interactions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to amutant PKD1 protein involves preparing a reaction mixture of the PKD1protein, which can be a mutant, including a variant, and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact, then isolating the interaction product (complex)or detecting the complex in the reaction mixture. Such assays can beconducted in a heterogeneous or homogeneous format. Heterogeneous assaysinvolve anchoring PKD1 or the test substance onto a solid phase anddetecting PKD1 test substance complexes anchored on the solid phase atthe end of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested.

In addition, methods suitable for detecting protein-protein interactionscan be employed for identifying novel PKD1 cellular or extracellularprotein interactions based upon the mutant or variant PKD1 polypeptidesof the invention. For example, some traditional methods that can beemployed are co-immunoprecipitation, crosslinking and copurificationthrough gradients or chromatographic columns. Additionally, methods thatresult in the simultaneous identification of the genes coding for theprotein interacting with a target protein can be employed. These methodsinclude, for example, probing expression libraries with labeled targetprotein, using this protein in a manner similar to antibody probing ofEgt libraries. One such method for detecting protein interactions invivo is the yeast two hybrid system. One version of this system has beendescribed (Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582, 1991)and can be performed using commercially available reagents (Clontech;Palo Alto Calif.).

A PKD1 polypeptide (e.g., a variant or mutant) of the invention caninteract with one or more cellular or extracellular proteins in vivo.Such cellular proteins are referred to herein as “binding partners”.Compounds that disrupt such interactions can be useful in regulating theactivity of a PKD1 polypeptide, especially mutant PKD1 polypeptides.Such compounds include, for example, molecules such as antibodies,peptides, peptidomimetics and the like.

In instances whereby ADPKD symptoms are associated with a mutationwithin the PKD1 polynucleotide (e.g., SEQ ID NO:1 having a mutation atT3110C; T8298G; A9164G; G9213A; C9326T; G10064A or the like; see Example2), which produces PKD1 polypeptides having aberrant activity, compoundsidentified that disrupt such activity can therefore inhibit the aberrantPKD1 activity and reduce or treat ADPKD1-associated symptoms or ADPKDdisease, respectively (see Table 4). For example, compounds can beidentified that disrupt the interaction of mutant PKD1 polypeptides withcellular or extracellular proteins, for example, the PKD2 gene product,but do not substantially effect the interactions of the normal PKD1protein. Such compounds can be identified by comparing the effectivenessof a compound to disrupt interactions in an assay containing normal PKD1protein to that of an assay containing mutant PKD1 polypeptide, forexample, a two hybrid assay.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the PKD1 protein, preferably amutant PKD1 protein, and its cellular or extracellular protein bindingpartner or partners involves preparing a reaction mixture containing thePKD1 protein and the binding partner under conditions and for a timesufficient to allow the two proteins to interact or bind, thus forming acomplex. In order to test a compound for inhibitory activity, reactionsare conducted in the presence or absence of the test compound, i.e., thetest compound can be initially included in the reaction mixture, oradded at a time subsequent to the addition of PKD1 and its cellular orextracellular binding partner; controls are incubated without the testcompound or with a placebo. The formation of any complexes between thePKD1 protein and the cellular or extracellular binding partner is thendetected. The formation of a complex or interaction in the controlreaction, but not in the reaction mixture containing the test compoundindicates that the compound interferes with the interaction of the PKD1protein and the binding partner. As noted above, complex formation orcomponent interaction within reaction mixtures containing the testcompound and normal PKD1 protein can also be compared to complexformation or component interaction within reaction mixtures containingthe test compound and mutant PKD1 protein. This comparison can beimportant in those cases wherein it is desirable to identify compoundsthat disrupt interactions of mutant but not normal PKD1 proteins.

Any of the binding compounds, including but not limited to, compoundssuch as those identified in the foregoing assay systems can be testedfor anti-ADPKD activity. ADPKD, an autosomal dominant disorder, caninvolve underexpression of a wild-type PKD1 allele, or expression of aPKD1 polypeptide that exhibits little or no PKD1 activity. In such aninstance, even though the PKD1 polypeptide is present, the overall levelof normal PKD1 polypeptide present is insufficient and leads to ADPKDsymptoms. As such increase in the level of expression of the normal PKD1polypeptide, to levels wherein ADPKD symptoms are ameliorated would beuseful. Additionally, the term can refer to an increase in the level ofnormal PKD1 activity in the cell, to levels wherein ADPKD symptoms areameliorated.

The identified compounds that inhibit PKD1 expression, synthesis and/oractivity can be administered to a patient at therapeutically effectivedoses to treat polycystic kidney disease. A therapeutically effectivedose refers to that amount of the compound sufficient to result inamelioration of symptoms of polycystic kidney disease. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.Additional factors that can be utilized to optimize dosage can include,for example, such factors as the severity of the ADPKD symptoms as wellas the age, weight and possible additional disorders that the patientcan also exhibit. Those skilled in the art will be able to determine theappropriate dose based on the above factors.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates can beformulated for administration by inhalation (either through the mouth orthe nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions can take the form of tablets or lozenges formulated inconventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin, for use in an inhaler can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. The compounds canalso be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

Alternatively, ADPKD can be caused by the production of an aberrantmutant form of the PKD1 protein, that either interferes with the normalallele product or introduces a novel function into the cell, which thenleads to the mutant phenotype. For example, a mutant PKD1 protein cancompete with the wild type protein for the binding of a substancerequired to relay a signal inside or outside of a cell.

Cell based and animal model based assays for the identification ofcompounds exhibiting anti-ADPKD activity are also encompassed within thepresent invention. Cells that contain and express mutant PKD1polynucleotide sequences (e.g., a sequence substantially identical tothe sequence as set forth in SEQ ID NO:1 and having one or moremutations of a C474T; G487A; T3110C; T8298G; A9164G; G9213A; C9326T;C9367T; G10064A; A10143G; T10234C; G10255T or the like; see Example 2),which encode a mutant PKD1 polypeptide, and thus exhibit cellularphenotypes associated with ADPKD, can be utilized to identify compoundsthat possess anti-ADPKD activity. Such cells can include cell linesconsisting of naturally occurring or engineered cells that expressmutant or express both normal and mutant PKD1 polypeptides. Such cellsinclude, but are not limited to renal epithelial cells, includingprimary and immortalized human renal tubular cells, MDCK cells, LLPCK1Cells, and human renal carcinoma cells. Methods of transforming cellwith PKD1 polynucleotide sequences encoding wild-type or mutant proteinsare described above.

Cells that exhibit ADPKD-like cellular phenotypes, can be exposed to acompound suspected of exhibiting anti-ADPKD activity at a sufficientconcentration and for a time sufficient to elicit an anti-ADPKD1activity in the exposed cells. After exposure, the cells are examined todetermine whether one or more of the ADPKD-like cellular phenotypes hasbeen altered to resemble a more wild type, non-ADPKD phenotype.

Among the cellular phenotypes that can be followed in the above assaysare differences in the apical/basolateral distribution of membraneproteins. For example, normal (i.e., non-ADPKD) renal tubular cells insitu and in culture under defined conditions have a characteristicpattern of apical/basolateral distribution of cell surface markers.ADPKD renal cells, by contrast, exhibit a distribution pattern thatreflects a partially reversed apical/basolateral polarity relative tothe normal distribution. For example, sodium-potassium ATPase generallyis found on the basolateral membranes of renal epithelial cells, butalso can be found on the apical surface of ADPKD epithelial cells, bothin cystic epithelia in vivo and in ADPKD cells in culture (Wilson etal., Am. J. Physiol. 260:F420-F430, 1991). Another marker that exhibitsan alteration in polarity in normal versus ADPKD affected cells is theEGF receptor, which is normally located basolaterally, but in ADPKDcells is mislocated to the apical surface. Such a apical/basolateralmarker distribution phenotype can be followed, for example, by standardimmunohistology techniques using antibodies specific to a markers ofinterest.

Assays for the function of PKD1 also can include a measure of the rateof cell growth or apoptosis, since dysregulation of epithelial cellgrowth can be a key step in cyst formation. The cysts are fluid filledstructures lined by epithelial cells that are both hyper-proliferativeand hyper-apoptotic (Evan et al., Kidney International 16:743-750, 1979;Kovacs and Gomba, Kidney Blood Press. Res. 21:325-328, 1998; Lanoix etal., Oncogene 13: 1153-1160, 1996; Woo, New Engl. J. Med. 333:18-25,1995, each of which is incorporated herein by reference). The cysticepithelium has a high mitotic rate in vivo as measured by PCNA staining(Nadasdy et al., J. Am. Soc. Nephrol. 5:1462-1468, 1995, which isincorporated herein by reference), and increased levels of expression ofother markers of proliferation (Klingel et al., Amer. J. Kidney Dis.19:22-30, 1992, which is incorporated herein by reference). In addition,cultured cells from ADPKD cystic kidneys have increased growth rates invitro (Wilson et al., Kidney Int. 30:371-380, 1986. Wilson, Amer. J.Kidney Dis. 17:634-637, 1991, each of which is incorporated herein byreference).

Further, in studies of rodent models of polycystic kidney disease, theepithelial cells that line cysts of animals with naturally occurringforms of PKD showed abnormalities similar to those reported in humanADPKD (Harding et al., 1992; Ramasubbu et al., J. Am. Soc. Nephrol.9:937-945, 1998; Rankin et al., J. Cell Physiol. 152:578-586, 1992;Rankin et al., In Vitro Cell Devel. Biol. Anim. 32:100-106, 1996, eachof which is incorporated herein by reference). Moreover, mice that havetransgenic overexpression of either c-myc or SV40-large T antigendeveloped PKD (Kelley et al. J. Am. Soc. Nephrol. 2:84-97, 1991; Trudelet al., Kidney Int. 39:665-671 1991, each of which is incorporatedherein by reference). Also, expression of recombinant full length PKD1in epithelial cells reduced their rate of growth and induced resistanceto apoptosis when challenged with stimuli such as serum starvation orexposure to UV light, which are known to stimulate apoptosis (Boletta etal., Mol. Cell. 6:1267-1273, 2000, which is incorporated herein byreference). As such, biochemical pathways that are activated by PKD1expression, including, for example, JAK2, STAT1/3, PI3 kinase, p21, andAKT, can provide surrogate markers for PKD1 activity.

The propensity of an epithelial cell to form tubules provides stillanother assay for the function of PKD1. In vivo, PKD is characterized bycystic transformation of renal tubules and pancreatic and biliaryductules. In vitro, expression of full length PKD1 induces spontaneoustubulogenesis in MDCK cells (Boletta et al., supra, 2000). In this modelsystem, control MDCK cells, which did not express recombinant wild typefull length PKD1, formed cystic structures unless treated withhepatocyte growth factor or with fibroblast conditioned medium whencultured suspended in collagen. In contrast, MDCK cells that expressedthe full length wild type recombinant form of PKD1 spontaneously formedtubules in the absence of exogenous factors when cultured in thismanner. As such, this model system can be used to identify ligands thatbind to and activate the PKD1 protein, to determine pathways that aretargeted for activation by therapeutic agents, and as an assay system toevaluate the effect of sequence variants on PKD1 function.

Additionally, assays for the function of a PKD1 polypeptide can, forexample, include a measure of extracellular matrix (ECM) components,such as proteoglycans, laminin, fibronectin and the like, in thatstudies in both ADPKD and in rat models of acquired cystic disease(Carone et al., Kidney International 35:1034-1040, 1989) have shownalterations in such components. Thus, any compound that serves to createan extracellular matrix environment that more fully mimics the normalECM should be considered as a candidate for testing for an ability toameliorate ADPKD symptoms.

In addition, it is contemplated that the present invention can be usedto measure the ability of a compound, such as those identified in theforegoing binding assays, to prevent or inhibit disease in animal modelsfor ADPKD. Several naturally-occurring mutations for renal cysticdisease have been found in animals, and are accepted in the art asmodels of ADPKD and provide test systems for assaying the effects ofcompounds that interact with PKD1 proteins. Of these models, theHan:SPRD rat model, provides an autosomal dominant model system (see,for example, Kaspareit-Rittinghausen et al., Vet. Path. 26:195, 1989),and several recessive models also are available (Reeders, NatureGenetics 1:235, 1992). In addition, knock-out mice, in which the PKD1 orPKD2 gene has been disrupted, are available and provide a relevant modelsystem for genetic forms of ADPKD. As such, the PKD1 and PKD2 knock-outmice can be useful for confirming the effectiveness in vivo of compoundsthat interact with PKD1 proteins in vitro (see, for example, Wu et al.,Nat. Genet. 24:75-78, 2000; Kim et al., Proc. Natl. Acad. Sci., USA97:1731-1736, 2000; Lu et al., Nat. Genet. 21:160-161, 1999; Wu et al.,Cell 93:177-188, 1998; Lu et al., Nat. Genet. 17:179-181, 1997, each ofwhich is incorporated herein by reference).

Animal models exhibiting ADPKD-like symptoms associated with one or moreof the mutant PKD1 polynucleotide sequences as disclosed herein can alsobe engineered by utilizing the PKD1 polynucleotide sequences such inconjunction with well known methods for producing transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, mini-pigs, goats, and non-human primates,e.g., baboons, squirrels, monkeys, and chimpanzees can be used togenerate such ADPKD animal models or transgenic animals. In instanceswhere the PKD1 mutation leading to ADPKD symptoms causes a drop in thelevel of PKD1 protein or causes an ineffective PKD1 protein to be made(e.g., the PKD1 mutation is a dominant loss-of-function mutation, suchas a W3001X, i.e., truncated after amino acid residue 3000, or a T3110Cmutation; see, also, Table 4) various strategies can be utilized togenerate animal models exhibiting ADPKD-like symptoms.

The present invention also provides transgenic non-human organisms,including invertebrates, vertebrates and mammals. For purposes of thesubject invention, these animals are referred to as “transgenic” whensuch animal has had a heterologous DNA sequence, or one or moreadditional DNA sequences normally endogenous to the animal (collectivelyreferred to herein as “transgenes”) chromosomally integrated into thegerm cells of the animal. The transgenic animal (including its progeny)will also have the transgene integrated into the chromosomes of somaticcells.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods can be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191.

In yet another such method, embryonic cells are infected with aretrovirus containing the transgene whereby the germ cells of the embryohave the transgene chromosomally integrated therein. When the animals tobe made transgenic are avian, because avian fertilized ova generally gothrough cell division for the first twenty hours in the oviduct,microinjection into the pronucleus of the fertilized egg is problematicdue to the inaccessibility of the pronucleus. Therefore, of the methodsto make transgenic animals described generally above, retrovirusinfection is preferred for avian species, for example as described inU.S. Pat. No. 5,162,215. If microinjection is to be used with avianspecies, however, the method of Love et al., (Biotechnology, 12, Jan.1994) can be utilized whereby the embryo is obtained from a sacrificedhen approximately two and one-half hours after the laying of theprevious laid egg, the transgene is microinjected into the cytoplasm ofthe germinal disc and the embryo is cultured in a host shell untilmaturity. When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ova canfirst be centrifuged to segregate the pronuclei for bettervisualization.

The non-human transgenic animals of the invention include, for example,bovine, porcine, ovine and avian animals (e.g., cow, pig, sheep,chicken, turkey). Such transgenic non-human animals are produced byintroducing a transgene into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for microinjection. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host gene before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal that includesexogenous genetic material within all of its cells. A transgenic animalcan be produced by cross-breeding two chimeric animals that includeexogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals that include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the invention,the transgene is digested and purified free from any vector DNA e.g. bygel electrophoresis. It is preferred that the transgene include anoperatively associated promoter that interacts with cellular proteinsinvolved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionein, skeletalactin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR,and thymidine kinase. Promoters for viral long terminal repeats (LTRs)such as Rous Sarcoma Virus can also be employed. When the animals to bemade transgenic are avian, preferred promoters include those for thechicken θ-globin gene, chicken lysozyme gene, and avian leukosis virus.Constructs useful in plasmid transfection of embryonic stem cells willemploy additional regulatory elements well known in the art such asenhancer elements to stimulate transcription, splice acceptors,termination and polyadenylation signals, and ribosome binding sites topermit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, Proc.Natl. Acad. Sci. USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan et al., In “Manipulating the Mouse Embryo” (Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. 1986)). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner et al.,Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten et al.,Proc. Natl. Acad. Sci. USA 82:6148-6152, 1985). Transfection is easilyand efficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (Van der Putten, supra; Stewart, et al., EMBO J.6:383-388, 1987). Alternatively, infection can be performed at a laterstage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623-628, 1982). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells that formed the transgenic nonhumananimal. Further, the founder can contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal., supra, 1982).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. Nature292:154-156, 1981; Bradley et al., Nature 309:255-258, 1984; Gossler etal., Proc. Natl. Acad. Sci. USA 83:9065-9069, 1986; and Robertson etal., Nature 322:445-448, 1986). Transgenes can be efficiently introducedinto the ES cells by DNA transfection or by retro virus-mediatedtransduction. Such transformed ES cells can thereafter be combined withblastocysts from a nonhuman animal. The ES cells thereafter colonize theembryo and contribute to the germ line of the resulting chimeric animal(for review see Jaenisch, Science 240:1468-1474, 1988).

The transgene can be any piece of DNA that is inserted by artifice intoa cell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) that developsfrom that cell. Such a transgene can include a gene that is partly orentirely heterologous (i.e., foreign) to the transgenic organism, or canrepresent a gene homologous to an endogenous gene of the organism.Included within this definition is a transgene created by the providingof an RNA sequence that is transcribed into DNA, then incorporated intothe genome. The transgenes of the invention include DNA sequences thatencode a mutant PKD1 polypeptide, for example, a polypeptide having anamino acid sequence substantially identical to SEQ ID NO:2 and having amutation of a A88V, a W967R, a L2696R, an R2985G, an R3039C, a V3285I, aH3311R, or any combination thereof; or encoding a truncated PKD1polypeptide ending at amino acid 3000 (also referred to herein as“W3001X”, where “X” indicates STOP codon; see, also, Table 4) andinclude sense, antisense, and dominant negative encodingpolynucleotides, which can be expressed in a transgenic non-humananimal. The term “transgenic” as used herein also includes any organismwhose genome has been altered by in vitro manipulation of the earlyembryo or fertilized egg or by any transgenic technology to induce aspecific gene knockout. The term “gene knockout” as used herein, refersto the targeted disruption of a gene in vivo with complete or partialloss of function that has been achieved by any transgenic technologyfamiliar to those in the art. In one embodiment, transgenic animalshaving a gene knockout are those in which the target gene has beenrendered nonfunctional by an insertion targeted to the gene to berendered non-functional by homologous recombination.

The invention also includes animals having heterozygous mutations in orpartial inhibition of function or expression of a PKD1 polypeptide. Oneof skill in the art would readily be able to determine if a particularmutation or if an antisense molecule was able to partially inhibit PKD1expression. For example, in vitro testing can be desirable initially bycomparison with wild-type (e.g., comparison of northern blots to examinea decrease in expression). After an embryo has been microinjected,colonized with transfected embryonic stem cells or infected with aretrovirus containing the transgene (except for practice of the subjectinvention in avian species, which is addressed elsewhere herein), theembryo is implanted into the oviduct of a pseudopregnant female. Theprogeny are tested for incorporation of the transgene by Southern blotanalysis of blood samples using transgene specific probes. PCR isparticularly useful in this regard. Positive progeny (P₀) are crossbredto produce offspring (P₁) that are analyzed for transgene expression bynorthern blot analysis of tissue samples.

In order to distinguish expression of like species transgenes fromexpression of an endogenous PKD1-related gene, a marker gene fragmentcan be included in the construct in the 3′ untranslated region of thetransgene and the northern blot probe designed to probe for the markergene fragment. The serum levels of a PKD1 polypeptide can also bemeasured in the transgenic animal to determine the level of PKD1expression. A method of creating a transgenic organism also can includemethods of inserting a transgene into, for example, an embryo of analready created transgenic organism, the organism being transgenic for adifferent unrelated gene or polypeptide.

Transgenic organisms of the invention are highly useful in theproduction of organisms for study of, for example, polycystic kidneydisease or PKD1-related diseases or disorders and in identifying agentsor drugs that inhibit or modulate polycystic kidney disease, PKD1associated disorders and inheritance. Expression of a mutant human PKD1polynucleotide can be assayed, for example, by standard northern blotanalysis, and the production of the mutant human PKD1 polypeptide can beassayed, for example, by detecting its presence using an antibodydirected against the mutant human PKD1 polypeptide. Those animals foundto express the mutant human PKD1 polypeptide can then be observed forthe development of ADPKD-like symptoms.

As discussed above, animal models of ADPKD can be produced byengineering animals containing mutations in a copy of an endogenous PKD1gene that correspond to mutations within the human PKD1 polynucleotide.Utilizing such a strategy, a PKD1 homologue can be identified and clonedfrom the animal of interest, using techniques such as those describedherein. One or more mutations can be engineered into such a PKD1homologue that correspond to mutations within the human PKD1polynucleotide, as discussed above (e.g., resulting in a mutation of theamino acid sequence as set forth in SEQ ID NO:2 and having a mutation ofa A88V, a W967R, a L2696R, an R2985G, a W3001X, an R3039C, a V3285I, aH3311R, or any combination thereof; see, also, Table 4). As disclosedherein, a mutant polypeptide produced by such an engineeredcorresponding PKD1 homologue can exhibit an aberrant PKD1 activity thatis substantially similar to that exhibited by a mutant human PKD1protein. The engineered PKD1 homologue can then be introduced into thegenome of the animal of interest, using techniques such as thosedescribed, above. Accordingly, any of the ADPKD animal models describedherein can be used to test compounds for an ability to ameliorate ADPKDsymptoms, including those associated with the expression of a mutantPKD1 polypeptide substantially identical to SEQ ID NO:2 and having themutation A88V, W967R, L2696R, R2985G, W3001X, R3039C, V3285I, H3311R, ora combination thereof (see Example 2 and Table 4).

As discussed above, mutations in the PKD1 polynucleotide that causeADPKD can produce a form of the PKD1 protein that exhibits an aberrantactivity that leads to the formation of ADPKD symptoms. A variety oftechniques can be utilized to inhibit the expression, synthesis, oractivity of such mutant PKD1 polynucleotides and polypeptides. Forexample, compounds such as those identified through assays described,above, which exhibit inhibitory activity, can be used in accordance withthe invention to ameliorate ADPKD symptoms. Such molecules can include,but are not limited, to small and large organic molecules, peptides, andantibodies. Further, antisense and ribozyme molecules that inhibitexpression of a PKD1 polynucleotide, (e.g., a mutant PKD1polynucleotide), can also be used to inhibit the aberrant PKD1 activity.Such techniques are described, below. In yet another embodiment, triplehelix molecules can be utilized in inhibiting aberrant PKD1 activity.

Among the compounds that can exhibit anti-ADPKD activity are antisense,ribozyme, and triple helix molecules. Such molecules can be designed toreduce or inhibit mutant PKD1 activity by modulating the expression orsynthesis of PKD1 polypeptides. Techniques for the production and use ofsuch molecules are well known to those of skill in the art.

Double stranded interfering RNA molecules are especially useful toinhibit expression of a target gene. For example, double stranded RNAmolecules can be injected into a target cell or organism to inhibitexpression of a gene and the resultant polypeptide's activity. It hasbeen found that such double stranded RNA molecules are more effective atinhibiting expression than either RNA strand alone (Fire et al., Nature,19:391 (6669): 806-11, 1998).

When a disorder is associated with abnormal expression of a PKD1polypeptide (e.g., overexpression, or expression of a mutated form ofthe protein), a therapeutic approach that directly interferes with thetranslation of a PKD1 polypeptide (e.g., a wild type, variant or mutantPKD1 polypeptide) is possible. Alternatively, similar methodology can beused to study gene activity. For example, antisense nucleic acid, doublestranded interfering RNA or ribozymes could be used to bind to a PKD1mRNA sequence or to cleave it. Antisense RNA or DNA molecules bindspecifically with a targeted gene's RNA message, interrupting theexpression of that gene's protein product. The antisense binds to themessenger RNA forming a double stranded molecule that cannot betranslated by the cell. Antisense oligonucleotides of about 15 to 25nucleotides are preferred since they are easily synthesized and have aninhibitory effect just like antisense RNA molecules. In addition,chemically reactive groups, such as iron-linkedethylenediaminetetraacetic acid (EDTA-Fe) can be attached to anantisense oligonucleotide, causing cleavage of the RNA at the site ofhybridization. Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of at least about 15 nucleotidesalso are preferred because they are less likely to cause problems whenintroduced into the target PKD1 polypeptide producing cell. The use ofantisense methods to inhibit the in vitro translation of genes is wellknown in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher et al., AntisenseRes. and Devel., 1:227, 1991; Helene, Anticancer Drug Design, 6:569,1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences that encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences that are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-base recognition sequences are preferable to shorter recognitionsequences. These and other uses of antisense and ribozymes methods toinhibit the in vivo translation of genes are known in the art (e.g., DeMesmaeker et al., Curr. Opin. Struct. Biol., 5:343, 1995; Gewirtz etal., Proc. Natl. Acad. Sci. USA, 93:3161, 1996b; Stein, Chem. and Biol.3:319, 1996).

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, which include the following sequence: GUA, GUU and GUC.Once identified, short RNA sequences of about 15 to 30 ribonucleotidescorresponding to the region of the target gene containing the cleavagesite can be evaluated for predicted structural features, such assecondary structure, that can render the oligonucleotide sequenceunsuitable. The suitability of candidate targets can also be evaluatedby testing their accessibility to hybridization with complementaryoligonucleotides, using ribonuclease protection assays.

It is possible that the antisense, ribozyme, or triple helix moleculesdescribed herein can reduce or inhibit the translation of mRNA producedby mutant PKD1 alleles of the invention. In order to ensure thatsubstantial normal levels of PKD1 activity are maintained in the cell,nucleic acid molecules that encode and express PKD1 polypeptidesexhibiting normal PKD1 activity can be introduced into cells that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments. Such sequences can be introduced via gene therapymethods such as those described, below. Alternatively, it can bepreferable to coadminister normal PKD1 protein into the cell or tissuein order to maintain the requisite level of cellular or tissue PKD1activity.

Antisense RNA and DNA molecules, ribozyme molecules and triple helixmolecules of the invention can be prepared by any method known in theart for the synthesis of DNA and RNA molecules. These include techniquesfor chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribonucleotide or deoxyribonucleotides to the 5′ or 3′ endor both of the molecule or the use of phosphorothioate or 2′-O-methylrather than phosphodiesterase linkages within theoligodeoxyribonucleotide backbone.

As discussed above, mutations in the PKD1 polynucleotide that causeADPKD can lower the level of expression of the PKD1 polynucleotide or;alternatively, can cause inactive or substantially inactive PKD1proteins to be produced. In either instance, the result is an overalllower level of normal PKD1 activity in the tissues or cells in whichPKD1 is normally expressed. This lower level of PKD1 activity, then,leads to ADPKD symptoms. Thus, such PKD1 mutations represent dominantloss-of-function mutations. For example, a polynucleotide having asequence as set forth in SEQ ID NO:1 and having a mutation of a G9213Aresults in early termination of PKD1.

For example, normal PKD1 protein, at a level sufficient to ameliorateADPKD symptoms can be administered to a patient exhibiting such symptomsor having a mutant PKD1 polynucleotide. Additionally, DNA sequencesencoding normal PKD1 protein can be directly administered to a patientexhibiting ADPKD symptoms or administered to prevent or reduce ADPKDsymptoms where they have been diagnosed as having a PKD1 mutationidentified herein but have not yet demonstrated symptoms. Suchadministration can be at a concentration sufficient to produce a levelof PKD1 protein such that ADPKD symptoms are ameliorated.

Further, subjects with these types of mutations can be treated by genereplacement therapy. A copy of the normal PKD1 polynucleotide can beinserted into cells, renal cells, for example, using viral or non-viralvectors that include, but are not limited to vectors derived from, forexample, retroviruses, vaccinia virus, adeno-associated virus, herpesviruses, bovine papilloma virus or non-viral vectors, such as plasmids.In addition, techniques frequently employed by those skilled in the artfor introducing DNA into mammalian cells can be utilized. For example,methods including but not limited to electroporation, DEAE-dextranmediated DNA transfer, DNA guns, liposomes, direct injection, and thelike can be utilized to transfer recombinant vectors into host cells.Alternatively, the DNA can be transferred into cells through conjugationto proteins that are normally targeted to the inside of a cell. Forexample, the DNA can be conjugated to viral proteins that normallytarget viral particles into the targeted host cell.

Administering the whole gene or polypeptide is not necessary to avoidthe appearance of ADPKD symptoms. The use of a “minigene” therapyapproach also can serve to ameliorate such ADPKD symptoms (see Ragot etal., Nature 3:647, 1993; Dunckley et al., Hum. Mol. Genet. 2:717-723,1993). A minigene system uses a portion of the PKD1 Coding region thatencodes a partial, yet active or substantially active PKD1 polypeptide.As used herein, “substantially active” means that the polypeptide servesto ameliorate ADPKD symptoms. Thus, the minigene system utilizes onlythat portion of the normal PKD1 polynucleotide that encodes a portion ofthe PKD1 polypeptide capable of ameliorating ADPKD symptoms, and can,therefore represent an effective and even more efficient ADPKD therapythan full-length gene therapy approaches. Such a minigene can beinserted into cells and utilized via the procedures described, above,for full-length gene replacement. The cells into which the PKD1 minigeneare to be introduced are, preferably, those cells, such as renal cells,which are affected by ADPKD. Alternatively, any suitable cell can betransfected with a PKD1 minigene so long as the minigene is expressed ina sustained, stable fashion and produces a polypeptide that amelioratesADPKD symptoms.

A therapeutic minigene for the amelioration of ADPKD symptoms cancomprise a nucleotide sequence that encodes at least one PKD1polypeptide peptide domain, particularly a domain having an amino acidsequence substantially identical to a peptide portion SEQ ID NO:2 andhaving a mutation as shown in Table 4, for example, an A88V, W967R,L2696R, R2985G, W3001X, R3039C, V3285I, or H3311R mutation. Minigenesthat encode such PKD1 polypeptides can be synthesized and/or engineeredusing the PKD1 polynucleotide sequence (SEQ ID NO:1).

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit can comprise a carrier meanscontaining one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. One of the container means cancomprise a probe that is or can be detectably labeled. Such probe can bean oligonucleotide comprising at least 10 contiguous nucleotides andhaving a sequence of a fragment of SEQ ID NO:1 including: nucleotide474, wherein nucleotide 474 is a T; nucleotide 487, wherein nucleotide487 is an A; nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide8298, wherein nucleotide 8298 is a G; nucleotide 9164, whereinnucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is anA; nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367,wherein nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide10064 is an A; nucleotide 10143, wherein nucleotide 10143 is a G;nucleotide 10234, wherein nucleotide 10234 is a C; or nucleotide 10255,wherein nucleotide 10255 is a T (see, also, Example 2).

A kit containing one or more oligonucleotide probes of the invention canbe useful, for example, for qualitatively identifying the presence ofmutant PKD1 polynucleotide sequences in a sample, as well as forquantifying the degree of binding of the probe for determining theoccurrence of specific strongly binding (hybridizing) sequences, thusindicating the likelihood for a subject having or predisposed to adisorder associated with PKD1. Where the kit utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing reagents for amplification of the target nucleicacid sequence. When it is desirable to amplify the mutant targetsequence, this can be accomplished using oligonucleotide primers, whichare based upon identification of the flanking regions contiguous withthe target nucleotide sequence. For example, primers such as thoselisted below in Tables 1 and 2 can be included in the kits of theinvention. The kit can also contain a container comprising a reportermeans such as an enzymatic, fluorescent, or radionuclide label, whichcan be bound to or incorporated into the oligonucleotide and canfacilitate identification of the oligonucleotide.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES

The present invention is based upon the use of widely spacedPKD1-specific anchor primers in long range PCR to generate 5 kb to 10 kbPKD1 polynucleotide segments. After appropriate dilution, the PCRproducts can be used as a template for mutation screening using any oneof a variety of methods. Accordingly, a number of mutants have beenidentified in families with PKD1-associated disorders.

Using a number of PKD1-specific primers, eight templates ranging in sizefrom about 0.3 to 5.8 kb were generated that span from the 5′untranslated region to intron 34 and cover all exons in the replicatedregion including exon 1 and exon 22 (Example 1). These reagents wereused to evaluate 47 Asian PKD1 families (Example 2). Variant nucleotidesequences were found throughout the PKD1 polynucleotide sequence.

Forty-one That and 6 Korean ADPKD families were studied. Samples from 50healthy That blood donors collected in blood banks served as normalcontrols. Genomic DNA was extracted from either fresh or frozen wholeblood that had been stored for up to five years using commerciallyavailable kits (Puregene, Gentra) or standard phenol-chloroform methods.For the N23HA and 145.19 cell lines (Cell 77:881-894, 1994; Germino etal., Am J. Hum. Genet. 46:925-933, 1990; Ceccherini et al., Proc. Natl.Acad. Sci. USA 89:104-108, 1992, each of which is incorporated herein byreference; see, also, Watnick et al., supra, 1997), genomic DNA wasisolated using the Puregene DNA isolation kit.

Example 1 Long Range Specific Templates

A two-part strategy was used to generate and validate PKD1-specificprimers that could be used to amplify the replicated portion of PKD1.The sequence of PKD1 (SEQ ID NO:1) was aligned with that of twohomologues present in GenBank (Accession Number AC002039) and identifiedpotential sequence differences. Candidate primers were designed suchthat the mismatches were positioned at or adjacent to the 3′ end of theoligonucleotide so as to maximize their specificity for PKD1.

The primers were tested for specificity using rodent-human somatic cellhybrids that either contained only human 16p13.3 and therefore, humanPKD1 (145.19, a radiation hybrid), or that lacked 16p13.3 and containedonly the human PKD1-homologues (N23HA). FIG. 2 presents a representativeexample of this approach using the primer pair, BPF6 and thePKD1-specific primer BPR6. This primer pair amplified a product of thecorrect length (4.5 kb) under the stated conditions only when totalhuman genomic DNA or 145.19 DNA is used as template. Similar resultswere obtained when BPR6 was used in combination with the non-specificprimer 28F to generate a much shorter product.

As a final control, the absence of amplified product was verified usingN23HA as template to confirm that the results obtained using total humangenomic DNA and 145.19 DNA were due to the specificity of the primer andnot the result of other causes (i.e., difference in quality of DNA orratio of human/rodent template). A primer specific for the homologues(BPR6HG) was designed that was positioned the same distance from BPF6 asBPR6 and used to amplify a specific band of the same size as thecorresponding PKD1-long range product. As predicted, a product of thecorrect size was amplified from both N23HA and total genomic DNA, butnot from 145.19.

A total of eight primer pairs can be used to generate a series oftemplates that range in size from about 0.3 kb to 5.8 kb and include allexons and their flanking intron sequences in the replicated portion ofPKD1 (exons 1 to 34). Table 1 summarizes the details for each productand includes the sequence of each primer, its respective position withinthe gene, its expected size, and the optimal annealing temperature andextension time for its amplification. FIG. 1 illustrates the relativeposition of each product with respect to the overall gene structure. Itshould be noted that exon 1 and its flanking sequences were particularlyproblematic to evaluate. Primer design was greatly limited by the highdegree of homology and extreme GC bias in the region. A combination ofwidely space primers (to generate a fragment considerably larger thanthe segment of interest) and the GC melt system were used to circumventthese obstacles.

Specific details concerning the primer sequences, annealing temperaturesand extension times used for each long-range (LR) template are providedin Table 1 (all sequences in Tables 1 and 2 are shown in 5′ to 3′orientation from left to right). Three hundred to 400 ng of genomic DNAwas used as template for each LR product, except for exon 1 (see below).The long range PCR amplification was performed as follows in a PerkinElmer 9600 thermal cycler: denaturation at 95° C. for 3 min followed by35 cycles of a two-step protocol that included denaturation at 95° C.for 20 sec followed by annealing and extension at a temperature and fora time specific for each primer pair (Table 1). A final extension at 72°C. for 10 min was included in each program. The total PCR volume was 50Tl using 4 U of rTth DNA polymerase XL (Cetus, Perkin Elmer) and a finalMgOAC₂ Concentration of 0.9 mM. A hot start protocol as recommended bythe manufacturer was used for the first cycle of amplification. For theexon 1 LR product (Tl), the LR was generated using 500 ng of genomicDNA. The long range PCR amplification was modified as follows:denaturation 95° C. for 1 min followed by 35 two-step cycles ofdenaturation at 95° C. for 30 sec followed by annealing and extension at69° C. for 7 min. The total PCR volume was 50 Tl using 1 Tl ofAdvantage-GC genomic polymerase (Clontech), GC melt of 1.5 M and finalMgOAC₂ concentration of 1.1 mM.

TABLE 1 Oligonucleotide Primers for Long-Range Specific Templatesfrom Exon 1-34 of PKD1 Gene Position Size Tm ET SEQ Template PrimersSequence 5′□3′ (5′) (kb) (° C.) (Min) ID NO: T1 BPF14*CCATCCACCTGCTGTGTGAC  2043 2.2 69 7  3 CTGGTAAAT BPR9CCACCTCATCGCCCCTTCCT  4290  4 AAGCAT T2-7 BPF9* ATTTTTTGAGATGGAGCTTC17907 4.6 68 7  5 ACTCTTGCAGG BPR4 CGCTCGGCAGGCCCCTAACC 22489  6 T8-12BPF12 CCGCCCCCAGGAGCCTAGAC 22218 4.2 68 7  7 G BPR5*CATCCTGTTCATCCGCTCCA 26363  8 CGGTTAC T13-15 F13 TGGAGGGAGGGACGCCAATC26246 4.4 68 7  9 R27* GTCAACGTGGGCCTCCAAGT 30612 10 T15-21 F26*AGCGCAACTACTTGGAGGCC 30603 3.4 70 4.5 11 C R2 GCAGGGTGAGCAGGTGGGGC 3395312 CATCCTAC T22 BPF15 GAGGCTGTGGGGGTCCAGTC 36815 0.3 72 1 13 AAGTGGBPR12* AGGGAGGCAGAGGAAAGGGC 37136 14 CGAAC T23-28 BPF6CCCCGTCCTCCCCGTCCTTT 37325 4.2 69 7 15 TGTC BPR6* AAGCGCAAAAGGGCTGCGTC41524 16 G T29-34 BPF13* GGCCCTCCCTGCCTTCTAGG 41504 5.8 68 8 17 CGKG8R25* GTTGCAGCCAAGCCCATGTT 47316 18 A Tm-annealing temperature;ET-extension time; *PKD1-specific primer. Bold type in BPR12 primersequence identifies intentional replacement of C by A to enhancediscrimination of PKD1 from homologs.

The long-range templates were serially diluted (1:10⁴ or 1:10⁵) toremove genomic contamination, then used as templates for nested PCR of200-400 bp exonic fragments. A total of 17 new primer pairs weredeveloped for exons 1-12 and exon 22. The sequences and PCR conditionsfor each new pair are summarized in Table 2. Primer sequences and PCRconditions for exons 13-21 and 23-34 are described in Watnick et al.,Am. J. Hum. Genet. 65:1561-1571, 1999; and Watnick et al., Hum. Mol.Genet. 6:1473-1481; 1997, which are incorporated herein by reference.Intron based primers were positioned approximately 30-50 bp away fromconsensus splice sites. Exons larger than approximately 400 bp weresplit into overlapping fragments of less than or equal to 350 bp. Two T1of diluted long range (LR) product was used as template foramplification of each exon. Single strand conformation analysis wasperformed using standard protocols. SSCA analysis was performed by useof 8% polyacrylamide gels with 5% glycerol added. The radiolabeled PCRproducts were diluted with loading buffer, were denatured by heating at95° C. for 5 min, then were placed on ice prior to being loaded and runon the gel at room temperature. Gels were run at 400 V overnight, dried,and placed on X-Omat XAR film (Kodak) at room temperature. Aberrantlymigrating bands detected by SSCA were cut from the gel and eluted into100 Tl of sterile water overnight. The eluted products were re-amplifiedusing the same set of primers, purified using Centricon-100 columns(Amicon) and then sequences.

TABLE 2 Nested Primers Used for Mutation Detection Fragment size T_(m)Exons Primer Primer Sequence 5′→3′ (bp) (° C.) SEQ ID NO: T1 1F1GGTCGCGCTGTGGCGAAGG 328 67  19 T1 1R1 CGGCGGGCGGCATCGT  20 T1 1F2ACGGCGGGGCCATGCG 348 67  21 T1 1R2 GCGTCCTGGCCCGCGTCC  22 T2-7 2FTTGGGGATGCTGGCAATGTG 272 62  23 T2-7 2R GGGATTCGGCAAAGCTGATG  24 T2-7 3FCCATCAGCTTTGCCGAATCC 171 62  25 T2-7 3R AGGGCAGAAGGGATATTGGG  26 T2-7 4FAGACCCTTCCCACCAGACCT 299 62  27 T2-7 4R TGAGCCCTGCCCAGTGTCT  28 T2-7 5F1GAGCCAGGAGGAGCAGAACCC 259 65  29 T2-7 5R1 AGAGGGACAGGCAGGCAAAGG  30 T2-75F2 CCCAGCCCTCCAGTGCCT 284 65  31 T2-7 5R2 CCCAGGCAGCACATAGCGAT  32 T2-75F3 CCGAGGTGGATGCCGCTG 294 65  33 T2-7 5R3 GAAGGGGAGTGGGCAGCAGAC  34T2-7 6F CACTGACCGTTGACACCCTCG 281 65  35 T2-7 6R TGCCCCAGTGCTTCAGAGATC 36 T2-7 7F GGAGTGCCCTGAGCCCCCT 311 65  37 T2-7 7R CCCCTAACCACAGCCAGCG 38 T8-12 8F TCTGTTCGTCCTGGTGTCCTG 215 65  39 T8-12 8RGCAGGAGGGCAGGTTGTAGAA  40 T8-12 9F GGTAGGGGGAGTCTGGGCTT 253 65  41 T8-129R GAGGCCACCCCGAGTCC  42 T8-12 10F GTTGGGCATCTCTGACGGTG 364 65  43 T8-1210R GGAAGGTGGCCTGAGGAGAT  44 T8-12 11F2 GGGGTCCACGGGCCATG 311 67  45T8-12 11R2 AAGCCCAGCAGCACGGTGAG  46 T8-12 11midF GCTTGCAGCCACGGAAC 38665  47 T8-12 11midR GCAGTGCTACCACTGAGAAC  48 T8-12 11F1TGCCCCTGGGAGACCAACGATAC 303 67  49 T8-12 11R1 GGCTGCTGCCCTCACTGGGAAG  5012 12F GAGGCGACAGGCTAAGGG 286 64  51 12R-2 CATGAAGCAGAGCAGAAGG  61 1313F: TGGAGGGAGGGACGCCAATC 308 67  62 13R: GAGGCTGGGGCTGGGACAA  63 1414F: CCCGGTTCACTCACTGCG 220 64  64 14R: CCGTGCTCAGAGCCTGAAAG  65 1515F16: CGGGTGGGGAGCAGGTGG 280 67  66 15R16: GCTCTGGGTCAGGACAGGGGA  67 1515F15: CGCCTGGGGGTGTTCTTT 270 64  68 15R15: ACGTGATGTTGTCGCCCG  69 1515F14: GCCCCCGTGGTGGTCAGC 250 67  70 15R14: CAGGCTGCGTGGGGATGC  71 1515F13: CTGGAGGTGCTGCGCGTT 256 67  72 15R13: CTGGCTCCACGCAGATGC  73 1515F12: CGTGAACAGGGCGCATTA 270 65  74 15R12: GCAGCAGAGATGTTGTTGGAC  75 1515F11: CCAGGCTCCTATCTTGTGACA 259 60  76 15R11: TGAAGTCACCTGTGCTGTTGT  7715 15F10: CTACCTGTGGGATCTGGGG 217 67  78 15R10: TGCTGAAGCTCACGCTCC  7915 15F9: GGGCTCGTCGTCAATGCAAG 267 67  80 15R9: CACCACCTGCAGCCCCTCTA  8115 15F8: 5CCGCCCAGGACAGCATCTTC 261 64  82 15R8: CGCTGCCCAGCATGTTGG  8315 15F7: CGGCAAAGGCTTCTCGCTC 288 64  84 15R7: CCGGGTGTGGGGAAGCTATG  8515 15F6: CGAGCCATTTACCACCCATAG 231 65  86 15R6: GCCCAGCACCAGCTCACAT  8715 15F5: CCACGGGCACCAATGTGAG 251 64  88 15R5: GGCAGCCAGCAGGATCTGAA  8915 15F4: CAGCAGCAAGGTGGTGGC 333 67  90 15R4: GCGTAGGCGACCCGAGAG  91 1515F3: ACGGGCACTGAGAGGAACTTC 206 64  92 15R3: ACCAGCGTGCGGTTCTCACT  93 1515F2: GCCGCGACGTCACCTACAC 265 67  94 15R2: TCGGCCCTGGGCTCATCT  95 1515F1: GTCGCCAGGGCAGGACACAG 228 68  96 R27′: AGGTCAACGTGGGCCTCCAA 113 1515F1-1: ACTTGGAGGCCCACGTTGACC 276 69  97 15R1-1: TGATGGGCACCAGGCGCTC  9815 15F1-2: CATCCAGGCCAATGTGACGGT 266 64  99 15R1-2:CCTGGTGGCAAGCTGGGTGTT 100 16 16F: TAAAACTGGATGGGGCTCTC 294 56 101 16R:GGCCTCCACCAGCACTAA 102 17 17F: GGGTCCCCCAGTCCTTCCAG 244 67 103 17R:TCCCCAGCCCGCCCACA 104 18 18F: GCCCCCTCACCACCCCTTCT 342 67 105 18R:TCCCGCTGCTCCCCCCAC 106 19 19F: GATGCCGTGGGGACCGTC 285 67 107 19R:GTGAGCAGGTGGCAGTCTCG 108 20 20F: CCACCCCCTCTGCTCGTAGGT 232 64 109 20R:GGTCCCAAGCACGCATGCA 110 21 21F: TGCCGGCCTCCTGCGCTGCTGA 232 67 111TWR2-1: GTAGGATGGCCCCACCTGCTCAC 112 CCTGC

Variants that were predicted to alter a restriction site were confirmedby restriction enzyme digestion analysis of re-amplified products. Incases where the change did not alter a restriction site, primers weredesigned with mismatches that create a new restriction site whencombined with the point mutation in question. The following primercombinations were utilized:

ASP1 + 26R (ASP1; 5′-CTGGTGACCTACATGGTCATGGCC GAGATC-3′; SEQ ID NO: 55);ASP2 + 30R (ASP2; 5′-GGTTGTCTATCCCGTCTACCTGGCCCTCCT-3′; SEQ ID NO: 56);ASP3 + 30F (ASP3; 5′-GTCCCCAGCCCCAGCCCACCTGGCC-3′; SEQ ID NO: 57).

When possible, segregation of the variant with the disease phenotype wastested. In cases where a missense change was unable to be determined onthe normal haplotype (and thus be a normal variant) the mutation wastested for in a panel of 50 normal controls.

Example 2 Mutation Screening

The new PKD1-specific products were generated from one affected memberof each of the 47 Asian families and then used as template for mutationdetection of exons 1-12 and 22-34. Table 2 (above) lists the sequenceand PCR condition for primer pairs that were used for nestedamplification of individual exons and their adjacent intronic sequence.Overlapping pairs were designed for segments>400 base pairs in length.

A total of 13 novel variants were detected by SSCA using the conditionsdescribed above. Two are highly likely to be pathogenic mutations, fourare predicted to encode missense substitutions not found in normals andseven are normal variants (see Table 3).

The first pathogenic mutation is a G to A transition at position 9213 inexon 25 that is predicted to result in a nonsense codon (W3001X). Itspresence was confirmed by restriction analysis using the enzyme Mae Iand it was found to segregate with disease. This variant is predicted totruncate the protein near the carboxyl end of the Receptor for Egg Jelly(REJ) domain. The W3001X mutation results in a greatly truncated productmissing all of the membrane spanning elements, intervening loops andcarboxy terminus. The second mutation (T3110C) is predicted to result ina non-conservative amino acid substitution (W967R) at a criticalposition of one of the PKD repeats. The mutation is unique to the familyin which it was found and was not observed in a screen of over 100normal That chromosomes. The W967R missense mutation is predicted todisrupt the secondary structure of PKD domain 3. The WDFGDGS (SEQ IDNO:58) motif within the CC′ loop region is the most conserved sequenceof the PKD domains. The tryptophan is replaced is the first residue ofthe turn at the end of the C strand and is conserved in 14 out of 16 PKDdomains. Moreover, it is evolutionarily conserved in mouse and Fugupolycystin-1.

TABLE 3 Mutations Identified in the PKD1 Gene in a Thai PopulationNucleic Acid Confirmation Patient Exon Change Codon Change ConsequenceEnzyme Pathogenic RAMA28-01⁰ 12 T3110C W967R Missense (disrupt BsaW 1(cut NC) PKD domain3) RAMA59-02* 25 G9213A W3001X Nonsense (early Mae Itermination) Variants not found in 100 chromosomes RAMA3-02* 22 T8298GL2696R Missense HinP1 I RAMA87-01* 25 A9164G R2985G Missense BsrB 1RAMA87-01* 25 C9326T R3039C Missense Fau I (cut NC) RAMA45-03* 29G10064A V3285I Missense Bsm I Probable normal variants RAMA7-06 2 C474TA88V Missense Hph I RAMA107-01 2 G487A A92A Silent change TspR IRAMA94-01 25 C9367T G3052G Silent change Sfo I (cut NC) RAMA66-01 30A10143G^(HG) H3311R Missense Nsp I (cut NC) RAMA66-01 30 T10234C^(HG)L3341L Silent change ASP1 + BseR I RAMA51-01 30 G10255T R3348R Silentchange ASP2 + MSC I *Segregation with disease; ⁰cannot test forsegregation; NC—Normal control; HG—Present in one copy of thehomologues; ASP—Allele-specific primer.

These pathogenic mutations add to previously identified pathogenicmutations, including a deletion of G3336 (−G3336) in exon 13, resultingin a frame shift after amino acid 1041 (FS1041); C4168T (Q1653)X),C6089T (Q1960X) and C6326T (Q2039X) mutations in exon 15, each resultingin a nonsense termination; −G7205-G7211 in exon 16, resulting in aFS2331; a C7415T (R2402X) mutation in exon 18, resulting in a nonsensetermination; a C7883T (Q2558X) mutation in exon 19, resulting in anonsense termination; and a −C8159-T8160 mutation in exon 21, resultingin a FS2649 (Phakdeekitcharoen et al., supra, 2000). In addition,probable pathogenic mutations including G3707A (G1166S) and T6078A(V1956E) missense mutations in exon 15, and a C7433T (R2408C) missensemutation and an insertion of a GCG trinucleotide between G7535 and G7536(extra Gly2422) in exon 18 have been identified (Phakdeekitcharoen etal., supra, 2000).

Four additional mutations unique to one of the families also wereidentified (see Table 3). The mutants segregate with disease, and werenot observed in a screen of over 100 normal That chromosomes. Three ofthe four variants are predicted to result in non-conservative amino acidsubstitutions. Two of them (A9164G, C9326T) are present in the sameallele of a single family (RAMA87). As such, these mutations meetseveral criteria expected of disease-producing mutations, including theyare not found in normal, ethnically matched chromosomes, they segregatewith the disease, and they result in non-conservative substitutions.

In one case a heteroduplex pattern was discovered for the exon 22product of the proband by standard agarose electrophoresis. Theheteroduplex pattern was confirmed to segregate with disease andsubsequently determined that the novel variant was the result of a T toG transversion at position 8298. This mutation is predicted tosubstitute arginine for leucine at position 2696 of the proteinsequence. This non-conservative substitution is within the REJ domain.Interestingly, the R3039C substitution occurs near a newly describedputative proteolytic cleavage site of polycystin-1,His(3047)-Leu-Thr-Ala(3050) (SEQ ID NO:59). In the correspondingposition of Fugu and murine polycystin-1, glutamic acid and arginine,respectively, are present, suggesting a non-critical role for anon-polar residue at this location.

Seven nucleotide substitutions that are likely normal variants were alsoidentified. Two are missense variants that do not segregate with diseasein the family in which they were discovered. The C474T substitutionresults in the conservative replacement of valine by alanine at position88 in the first leucine rich (LRR) repeat. The amino acid is notconserved between species and is not predicted to disrupt the LRRstructure. The second missense variant, A10143G, substitutes argininefor histidine at position 3311 within the first extracellular loopbetween TM2 and TM3. It too, is a conservative change involving aresidue whose identity is not evolutionarily conserved at this position.The other five variants were silent nucleotide substitutions that wereunique to the pedigree in which they were found and not found in morethan 100 normal chromosomes. It is possible that these variants can bepathogenic by affecting gene splicing in the region. Two of the normalvariants of exon 30, A10143G (H3311R) and T10234C (L3341L), wereclustered together in a single PKD1 haplotype. Interestingly, bothvariants also are present in at least one of the homologues, suggestinga previous gene conversion event as the original of these PKD1 variants.Additional PKD1 variants, which do not appear to be associated with aPKD1-associated disorder, include two silent mutations, G4885A (T1558T)and C6058T (S1949S), and a missense mutation, G6195A (R1995H), in exon15; a silent T7376C (L2389L) mutation in exon 17; a silent C7696T(C2495C) mutation in exon 18; and a missense G8021A (D2604N) mutation inexon 20 (Phakdeekitcharoen et al., supra, 2000).

Table 4 summarizes the clinical findings for the probands of 17 Thatfamilies. The genotypes and phenotypes for patients with ADPKD areshown. It has been estimated on the basis of studies of Caucasianpopulations that approximately 15% of mutations are localized to thenonreplicated portion of the PKD1 gene. If the same frequency is truefor the That population (the patients were not screened for mutations inthe nonreiterated portion), then the present studies have identifiedapproximately 45% to 54 percent of all mutations present in thenonreplicated region. This detection rate likely can be increased byusing more sensitive detection methods such as DHPLC (Kristensen et al.,supra, 2001), HTCSGD (Leung et al., supra, 2001), or the like.

TABLE 4 Genotypes and Phenotypes in Thai ADPKD1 Phenotype Renal Genotypeinsuff. Renal Palpable Liver Heart Valv. Brain Patients Age Exon CodonChange Consequence HT (Cr > 2) stone kidneys Cyst Abnorm. Aneur Ref.RAMA28-0 30 12 W967R Missense + − − − − − − RAMA103- 57 13 FS after 1041Frameshift + − + − + − − (*) RAMA49-0 26 15 G1166S Missense + − − + − −− (*) RAMA36-0 47 15 Q1653X Nonsense + − − + − − (*) RAMA108- 57 15V1956E Missense + + − − − − (*) RAMA77-0 53 15 Q1960X Nonsense + + − + +− − (*) RAMA32-0 36 15 Q2039X Nonsense + + − + − − (*) RAMA97-0 45 17R2402X Nonsense + + − − − − (*) RAMA96-0 30 18 R2408C Missense − − + − +− − (*) RAMA99-0 56 18 R2430X Nonsense + + + − + − − (*) RAMA66-0 39 182442 add'l. Gly Extra Glycine + − + − − − − (*) RAMA55-0 52 19 Q2558XNonsense − − − + + − − (*) RAMA5-01 53 21 FS after 2649Frameshift + + + + + + + (*) RAMA3-02 40 22 L2696R Missense + + − + + −− RAMA87-0 61 25 R2985G Missense − − − + + − − 25 R3039C Missense − −RAMA59-0 35 25 W3001X Nonsense + + + − − − − RAMA45-0 59 29 V32851Missense + + − − − − − HT—hypertension; Renal insuff.—renalinsufficiency; Heart Valv. Abnorm.—heart valvular abnormalities; BrainAneur.—brain aneurisyms; (*) Phakdeekitcharoen et al., supra, 2000.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method of amplifying a PKD1-specificpolynucleotide, comprising: (a) contacting a PKD1 polynucleotide in abiological sample obtained from a human with a primer that selectivelyhybridizes to a nucleotide sequence within a polycystic kidneydisease-associated protein-1 (PKD1) gene sequence to produce aPKD1-specific amplification product; and (b) amplifying the PKD1polynucleotide to obtain a PKD1-specific amplification product, whereinthe primer selectively hybridizes to a PKD1 gene sequence as set forthin SEQ ID NO:1, and wherein the primer is selected from the groupconsisting of SEQ ID NOs: 3, 5, 8, 14, 16 and
 17. 2. The method of claim1, wherein amplifying the PKD1 polynucleotide to obtain a PKD1-specificamplification product comprises the use of a primer pair selected fromSEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ IDNOS:13 and 14; SEQ ID NOS:15 and 16; and SEQ ID NOS:17 and
 18. 3. Themethod of claim 1, further comprising performing a nested PCR reactionby contacting the PKD1-specific amplification product with a primer setunder conditions suitable to perform nested PCR to obtain a nested PCRproduct.
 4. The method of claim 3, further comprising a dilution step,to remove genomic contamination from the amplification products, priorto the nested PCR reaction.
 5. The method of claim 3, wherein the primerset comprises one or more oligonucleotide selected from the groupconsisting of SEQ ID NOs: 28, 43, 48, 51, 60, 61, 63, 100 and
 102. 6.The method of claim 1, wherein amplifying the PKD1 polynucleotide toobtain a PKD1-specific amplification product comprises the use of one ormore oligonucleotides selected from the group consisting of SEQ ID NOs:6, 14 and 17.